Anti-integrin β1 antibody compositions and methods of use thereof

ABSTRACT

The current invention provides human variable chain framework regions and humanized antibodies comprising the framework regions, the antibodies being specific for integrin β1. The invention also provides methods for utilizing the antibodies, for example to treat diseases such as cancer.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application claims the benefit under 35 USC § 119(e) to U.S.Application Ser. No. 61/746,023, filed Dec. 26, 2012. The disclosure ofthe prior application is considered part of and is incorporated byreference in its entirety in the disclosure of this application.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates generally to the field of immunology, andmore specifically to anti-integrin antibodies and methods of usethereof.

Background Information

Cancer is one of the leading causes of death in the developed world,resulting in over 500,000 deaths per year in the United States alone.Over one million people are diagnosed with cancer in the U.S. each year,and overall it is estimated that more than 1 in 3 people will developsome form of cancer during their lifetime. Though there are more than200 different types of cancer, four of them including breast, lung,colorectal, and prostate, account for over half of all new cases.

Breast cancer is the most common cancer in women, with an estimate 12%of women at risk of developing the disease during their lifetime.Although mortality rates have decreased due to earlier detection andimproved treatments, breast cancer remains a leading cause of death inmiddle-aged women. Furthermore, metastatic breast cancer is still anincurable disease. On presentation, most patients with metastatic breastcancer have only one or two organ systems affected, but as the diseaseprogresses, multiple sites usually become involved. The most commonsites of metastatic involvement are locoregional recurrences in the skinand soft tissues of the chest wall, as well as in axilla andsupraclavicular areas. The most common site for distant metastasis isthe bone (30-40% of distant metastasis), followed by the lungs andliver. Although only approximately 1-5% of women with newly diagnosedbreast cancer have distant metastasis at the time of diagnosis,approximately 50% of patients with local disease eventually relapse withmetastasis within five years. At present the median survival from themanifestation of distant metastases is about three years.

Current methods of diagnosing and staging breast cancer include thetumor-node-metastasis (TNM) system that relies on tumor size, tumorpresence in lymph nodes, and the presence of distant metastases asdescribed in the American Joint Committee on Cancer: AJCC Cancer StagingManual. Philadelphia, Pa.: Lippincott-Raven Publishers, 5th ed., 1997,pp 171-180, and in Harris, J R: “Staging of breast carcinoma” in Harris,J. R., Hellman, S., Henderson, I. C, Kinne D. W. (eds.): BreastDiseases. Philadelphia, Lippincott, 1991. These parameters are used toprovide a prognosis and select an appropriate therapy. The morphologicappearance of the tumor may also be assessed but because tumors withsimilar histopathologic appearance can exhibit significant clinicalvariability, this approach has serious limitations. Finally, assays forcell surface markers can be used to divide certain tumors types intosubclasses. For example, one factor considered in the prognosis andtreatment of breast cancer is the presence of the estrogen receptor (ER)as ER-positive breast cancers typically respond more readily to hormonaltherapies such as tamoxifen or aromatase inhibitors than ER-negativetumors. Yet these analyses, though useful, are only partially predictiveof the clinical behavior of breast tumors, and there is much phenotypicdiversity present in breast cancers that current diagnostic tools failto detect and current therapies fail to treat.

Prostate cancer is the most common cancer in men in the developed world,representing an estimated 33% of all new cancer cases in the U.S., andis the second most frequent cause of death. Since the introduction ofthe prostate specific antigen (PSA) blood test, early detection ofprostate cancer has dramatically improved survival rates, and the fiveyear survival rate for patients with local and regional stage prostatecancers at the time of diagnosis is nearing 100%. Yet more than 50% ofpatients will eventually develop locally advanced or metastatic disease.

Currently radical prostatectomy and radiation therapy provide curativetreatment for the majority of localized prostate tumors. However,therapeutic options are very limited for advanced cases. For metastaticdisease, androgen ablation with luteinising hormone-releasing hormone(LHRH) agonist alone or in combination with anti-androgens is thestandard treatment. Yet despite maximal androgen blockage, the diseasenearly always progresses with the majority developingandrogen-independent disease. At present there is no uniformly acceptedtreatment for hormone refractory prostate cancer, and chemotherapeuticregimes are commonly used.

Lung cancer is the most common cancer worldwide, the third most commonlydiagnosed cancer in the United States, and by far the most frequentcause of cancer deaths. Cigarette smoking is believed responsible for anestimated 87% of all lung cancers making it the most deadly preventabledisease. Lung cancer is divided into two major types that account forover 90% of all lung cancers: small cell lung cancer (SCLC) andnon-small cell lung cancer (NSCLC). SCLC accounts for 15-20% of casesand is characterized by its origin in large central airways andhistological composition of sheets of small cells with little cytoplasm.SCLC is more aggressive than NSCLC, growing rapidly and metastasizingearly and often. NSCLC accounts for 80-85% of all cases and is furtherdivided into three major subtypes based on histology: adenocarcinoma,squamous cell carcinoma (epidermoid carcinoma), and large cellundifferentiated carcinoma.

Lung cancer typically presents late in its course, and thus has a mediansurvival of only 6-12 months after diagnosis and an overall 5 yearsurvival rate of only 5-10%. Although surgery offers the best chance ofa cure, only a small fraction of lung cancer patients are eligible withthe majority relying on chemotherapy and radiotherapy. Despite attemptsto manipulate the timing and dose intensity of these therapies, survivalrates have increased little over the last 15 years.

Colorectal cancer is the third most common cancer and the fourth mostfrequent cause of cancer deaths worldwide. Approximately 5-10% of allcolorectal cancers are hereditary with one of the main forms beingfamilial adenomatous polyposis (FAP), an autosomal dominant disease inwhich about 80% of affected individuals contain a germline mutation inthe adenomatous polyposis coli (APC) gene. Colorectal carcinoma has atendency to invade locally by circumferential growth and elsewhere bylymphatic, hematogenous, transperitoneal, and perineural spread. Themost common site of extralymphatic involvement is the liver, with thelungs the most frequently affected extra-abdominal organ. Other sites ofhematogenous spread include the bones, kidneys, adrenal glands, andbrain.

The current staging system for colorectal cancer is based on the degreeof tumor penetration through the bowel wall and the presence or absenceof nodal involvement. This staging system is defined by three majorDuke's classifications: Duke's A disease is confined to submucosa layersof colon or rectum; Duke's B disease has tumors that invade throughmuscularis propria and can penetrate the wall of the colon or rectum;and Duke's C disease includes any degree of bowel wall invasion withregional lymph node metastasis. While surgical resection is highlyeffective for early stage colorectal cancers, providing cure rates of95% in Duke's A patients, the rate is reduced to 75% in Duke's Bpatients and the presence of positive lymph node in Duke's C diseasepredicts a 60% likelihood of recurrence within five years. Treatment ofDuke's C patients with a post surgical course of chemotherapy reducesthe recurrence rate to 40%-50%, and is now the standard of care forthese patients.

Epithelial carcinomas of the head and neck arise from the mucosalsurfaces in the head and neck area and are typically squamous cell inorigin. This category includes tumors of the paranasal sinuses, the oralcavity, and the nasopharynx, oropharynx, hypopharynx, and larynx.

The annual number of new cases of head and neck cancers in the UnitedStates is approximately 40,000 per year, accounting for about 5 percentof adult malignancies. Head and neck cancers are more common in someother countries, and the worldwide incidence probably exceeds half amillion cases annually. In North American and Europe, the tumors usuallyarise from the oral cavity, oropharynx, or larynx, whereas nasopharynealcancer is more common in the Mediterranean countries and in the FarEast.

Traditional modes of therapy (radiation therapy, chemotherapy, andhormonal therapy), while useful, have been limited by the emergence oftreatment-resistant cancer cells. Clearly, new approaches are needed toidentify targets for treating head and neck cancer and cancer generally.

Pancreatic cancer is a malignant neoplasm originating from transformedcells arising in tissues forming the pancreas. The most common type ofpancreatic cancer, accounting for 95% of these tumors, is adenocarcinoma(tumors exhibiting glandular architecture on light microscopy) arisingwithin the exocrine component of the pancreas. A minority arise fromislet cells, and are classified as neuroendocrine tumors. Pancreaticcancer is the fourth most common cause of cancer-related deaths in theUnited States and the eighth worldwide.

Glioblastoma multiforme (GBM) is the most common malignant brain tumorin adults with a median survival of less than one year with maximaltherapy. To date, only three drugs have been approved by the FDA for GBMtreatment and overall survival has not improved in over 25 years.

Integrins are cell-adhesion molecules that are responsible formechanosensing the microenvironment and eliciting extracellular-matrix(ECM)-induced signaling in both normal and pathological states such asinflammation and cancer. Importantly, integrins lie at the interface ofthe cell and microenvironment, playing a key role in tumor progressionand regulating growth and survival pathways. Upregulation of many typesof integrins has been associated with epithelial malignancies,particularly during the processes of invasion, metastasis, andangiogenesis. Importantly, β1 integrins which coordinate much broaderfunctional activities such as inflammation, proliferation, adhesion, andinvasion have recently been implicated in therapeutic resistance inmultiple solid cancer models and hematopoietic malignancies.Importantly, this β1 integrin mediated resistance is thought to occur atthe level of the tumor cells themselves. In addition to the above, β1integrin has important functions during tumor vascularization such asVEGF-dependent and VEGF-independent angiogenesis by promoting migrationof vascular endothelial cells Inhibition of β1 integrin overcomesresistance to antiangiogenesis therapy via multiple potentialmechanisms: (1) preventing vessel cooption (and/or growth/invasion uponany classical ECM substrate; (2) reducing viability of tumor cells afterinsults such as ionizing radiation; (3) directly inhibiting tumor cellproliferation; (4) directly inhibiting the vascularization process; and(5) inhibiting the aggressive mesenchymal phenotype, includingspheroidal growth, typically seen after the establishment of therapyresistance.

Anti-integrin β1 compositions, such as integrin β1 targeted antibodiesmay also be important in for immunological/inflammatory diseases anddisorders given the role of integrin β1 in broader functional activitiesas discussed above. Further, diseases and disorders which may betargeted through the integrin β1 pathway include multiple sclerosis,Crohn's disease, rheumatoid arthritis, inflammatory bowel disease andthe like. Similarly, it is to be expected that certain eye relateddiseases may be targeted including wet age-related macular degeneration(AMD).

SUMMARY OF THE INVENTION

The present invention is based on the generation of humanized antibodieshaving human framework sequences which specifically bind integrin β1.Integrin β1 is known to be a protein over-expressed in solid tumors, andthus as a cancer cell marker useful in the characterization, study,diagnosis, and treatment of cancer. In addition, integrin β1 has beendemonstrated to drive cancer resistance to conventional (e.g.,chemotherapy and ionizing radiation) and targeted (e.g., trastuzumab,bevicizumab, lapatinib) therapies by orchestrating growth and survivalsignals from the tumor microenvironment. Humanization efforts have beenlimited in the past by the number of human frameworks available. Thisinvention meets the need for additional human framework sequences for anantibody the specifically binds Integrin β1.

In one embodiment, the present invention provides a humanized antibodywhich specifically binds integrin β1. The antibody includes a heavychain variable (VH) region and a light chain variable (VL) region,wherein the VH region has less than about 97%, 96% or 95% identity to aVH region having an amino acid sequence as set forth in SEQ ID NO:2, andwherein the VL region has less than about 97%, 96% or 95% identity to aVL region having an amino acid sequence as set forth in SEQ ID NO:4.

In another embodiment, the VH region has more than 75%, 80%, 85%, 90%,95% or 99% identity to a VH region having an amino acid sequence as setforth in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NOs:29-43 and SEQ ID NOs:91-100. In embodiments the VLregion has more than 75%, 80%, 85%, 90%, 95% or 99% identity to a VLregion having an amino acid sequence as set forth in SEQ ID NO:16, SEQID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NOs:44-57 and SEQ IDNOs:107-116.

In another embodiment, the antibody has CDRs of the VH and VL regionsfrom a donor antibody, such as OS2966. In embodiments, the CDRs of theVH region have amino acid sequences as set forth in SEQ ID NO:23, SEQ IDNO:24 and SEQ ID NO:25, and the CDRs of the VL region have amino acidsequences as set forth in SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28.

In one aspect, the invention provides an antibody which includes VH andVL regions of the present invention.

In another aspect, the invention provides a nucleic acid moleculeencoding the antibody of the present invention.

In another aspect, the invention provides a vector which includes anucleic acid molecule of the present invention.

In another aspect, the invention provides an isolated host cell whichincludes the vector of the present invention.

In another aspect, the invention provides pharmaceutical compositions.In one embodiment the pharmaceutical composition includes the antibodyof the present invention and a pharmaceutically acceptable carrier. Inone embodiment the pharmaceutical composition includes the nucleic acidmolecule of the present invention and a pharmaceutically acceptablecarrier.

In another aspect, the invention provides an immunoconjugate includingthe antibody of the present invention linked to detection or therapeuticmoiety.

In another aspect, the invention provides a chimeric protein includingthe antibody of the present invention operably linked to a separatepeptide, such as a cytokine.

In another embodiment, the invention provides a method of treating adisease in a subject. In embodiments, the method includes administeringto the subject the antibody of the present invention, the nucleic acidmolecule of the present invention, the pharmaceutical composition of thepresent invention, the immunoconjugate of the present invention, or thechimeric protein of the present invention, thereby treating the disease.In one embodiment the disease is a cell proliferative disorder, such ascancer.

In another embodiment, the invention provides a method of detecting adisease in a subject. The method includes contacting the antibody orimmunoconjugate of the present invention with a sample from the subject;detecting the level of integrin β1 via specific binding with integrinβ1; and comparing the detected level of integrin β1 to that of integrinβ1 in a normal sample, wherein an increased level of integrin β1 in thesample from the subject as compared to the normal sample is indicativeof a disease.

In another embodiment, the invention provides a VH region having morethan 75%, 80%, 85%, 90%, 95% or 99% identity to a VH region having anamino acid sequence as set forth in SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NOs:29-43 and SEQ IDNOs:91-100.

In another embodiment, the invention provides a VL region having morethan 75%, 80%, 85%, 90%, 95% or 99% identity to a VL region having anamino acid sequence as set forth in SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NOs:44-57 and SEQ ID NOs:107-116.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a pictorial representation depicting nucleic acid and aminoacid sequences of the VH region of antibody OS2966 produced by hybridomaOS2966.

FIG. 2 is a pictorial representation depicting nucleic acid and aminoacid sequences of the VL region of antibody OS2966 produced by hybridomaOS2966.

FIG. 3 is a pictorial representation depicting analysis of CDRs ofantibody OS2966 produced by hybridoma OS2966 which are utilized in theVH and VL regions of the antibody of the present invention.

FIG. 4 is a pictorial representation depicting the amino acid sequencesof CDRs of antibody OS2966 produced by hybridoma which are utilized inthe VH and VL regions of the antibody of the present invention.

FIG. 5 is a pictorial representation depicting nucleic acid and aminoacid sequences of the VH region of the antibody of the present inventionin one embodiment. Conserved CDRs are underlined.

FIG. 6 is a pictorial representation depicting nucleic acid and aminoacid sequences of the VH region of the antibody of the present inventionin one embodiment. Conserved CDRs are underlined.

FIG. 7 is a pictorial representation depicting nucleic acid and aminoacid sequences of the VH region of the antibody of the present inventionin one embodiment. Conserved CDRs are underlined.

FIG. 8 is a pictorial representation depicting nucleic acid and aminoacid sequences of the VH region of the antibody of the present inventionin one embodiment. Conserved CDRs are underlined.

FIG. 9 is a pictorial representation depicting nucleic acid and aminoacid sequences of the VH region of the antibody of the present inventionin one embodiment. Conserved CDRs are underlined.

FIG. 10 is a pictorial representation depicting nucleic acid and aminoacid sequences of the VL region of the antibody of the present inventionin one embodiment. Conserved CDRs are underlined.

FIG. 11 is a pictorial representation depicting nucleic acid and aminoacid sequences of the VL region of the antibody of the present inventionin one embodiment. Conserved CDRs are underlined.

FIG. 12 is a pictorial representation depicting nucleic acid and aminoacid sequences of the VL region of the antibody of the present inventionin one embodiment. Conserved CDRs are underlined.

FIG. 13 is a pictorial representation depicting nucleic acid and aminoacid sequences of the VL region of the antibody of the present inventionin one embodiment. Conserved CDRs are underlined.

FIG. 14 is a schematic diagram of vectors.

FIG. 15 is a pictorial representation depicting sequence analysis andalignment of humanized VH chains of the invention with comparison to VHof antibody OS2966 (SEQ ID NO:2). Sequences of FIG. 15 are as follows:OS2966 is SEQ ID NO:2; Variant 1 is SEQ ID NO:6; Variant 2 is SEQ IDNO:8; Variant 3 is SEQ ID NO:10; Variant 4 is SEQ ID NO:12; Variant 5 isSEQ ID NO:14; Variant 6 is SEQ ID NO:29; Variant 7 is SEQ ID NO:30;Variant 8 is SEQ ID NO:31; Variant 9 is SEQ ID NO:32; Variant 10 is SEQID NO:33; Variant 11 is SEQ ID NO:34; Variant 12 is SEQ ID NO:35;Variant 13 is SEQ ID NO:36; Variant 14 is SEQ ID NO:37; Variant 15 isSEQ ID NO:38; VH2 is SEQ ID NO:91; Variant 16 is SEQ ID NO:92; VH4 isSEQ ID NO:93; Variant 17 is SEQ ID NO:94; VH5 is SEQ ID NO:95; Variant18 is SEQ ID NO:96; VH6 is SEQ ID NO:97; Variant 19 is SEQ ID NO:98; VH7is SEQ ID NO:99; and Variant 20 is SEQ ID NO:100.

FIG. 16 is a pictorial representation depicting sequence analysis andalignment of humanized J regions (VH) of the invention corresponding toalignments shown in FIG. 15. Sequences are as follows: J1 is SEQ IDNO:101; J2 is SEQ ID NO:102; J3 is SEQ ID NO:103; J4 is SEQ ID NO:104;J5 is SEQ ID NO:105; and J6 is SEQ ID NO:106.

FIG. 17 is a pictorial representation depicting sequence analysis andalignment of humanized VL chains of the invention with comparison to VLof antibody OS2966 (SEQ ID NO:4). Sequences are as follows: OS2966 isSEQ ID NO:4; Variant 1 is SEQ ID NO:16; Variant 2 is SEQ ID NO:18;Variant 3 is SEQ ID NO:20; Variant 4 is SEQ ID NO:22; Variant 5 is SEQID NO:44; Variant 6 is SEQ ID NO:45; Variant 7 is SEQ ID NO:46; Variant8 is SEQ ID NO:47; Variant 9 is SEQ ID NO:48; Variant 10 is SEQ IDNO:49; Variant 11 is SEQ ID NO:50; Variant 12 is SEQ ID NO:51; Variant13 is SEQ ID NO:52; VkI is SEQ ID NO:107; Variant 14 is SEQ ID NO:108;VkII is SEQ ID NO:109; Variant 15 is SEQ ID NO:110; VkIII is SEQ IDNO:111; Variant 16 is SEQ ID NO:112; VkIV is SEQ ID NO:113; Variant 17is SEQ ID NO:114; VkVI is SEQ ID NO:115; and Variant 18 is SEQ IDNO:116.

FIG. 18 is a pictorial representation depicting sequence analysis andalignment of humanized J regions (kappa light chains) of the inventioncorresponding to alignments shown in FIG. 17. Sequences are as follows:J1 is SEQ ID NO:117; J2 is SEQ ID NO:118; J3 is SEQ ID NO:119; J4 is SEQID NO:120; and J5 is SEQ ID NO:121.

FIG. 19 is a pictorial representation depicting codon usage of the VHand VL chains of the present invention.

FIG. 20 is a graphical representation depicting relative affinity ofcomposite human antibody variants of the present invention (those shownin Table 2).

FIG. 21 is a graphical representation depicting relative affinity ofcomposite human antibody variants of the present invention (those shownin Table 2).

FIG. 22 is a graphical representation depicting relative affinity ofcomposite human antibody variants of the present invention (those shownin Table 2).

FIG. 23 is a graphical representation depicting relative affinity ofcomposite human antibody variants of the present invention (those shownin Table 2).

FIG. 24 is a graphical representation depicting relative affinity ofcomposite human antibody variants of the present invention (those shownin Table 2).

FIGS. 25A-D is a series of graphical representations illustratingfunctional validation in extracellular matrix (ECM) adhesion assays ofcomposite human antibody variants of the present invention. Functionalinhibition of the integrin β1 subunit with composite human antibodyvariants of the present invention (from Table 2) was assessed in anadhesion microplate assay on multiple ECM and in multiple cancer celllines. FIG. 25A utilizes PANC-1 human pancreatic cancer. FIG. 25Butilizes PANC-1 human pancreatic cancer. FIG. 25B utilizes PANC-1 humanpancreatic cancer. FIG. 25C utilized MDA-MB-231 human triple negativebreast cancer. FIG. 25D utilizes AsPC-1 human pancreatic cancer.

FIGS. 26A-B is a series of pictorial and graphical representationsillustrating functional validation in extracellular matrix (ECM)migration assays of composite human antibody variants of the presentinvention. Functional inhibition of the integrin β1 subunit withcomposite human antibody variants of the present invention (from Table2) was assessed in a microplate “scratch wound” migration assay on ECMcomponent fibronectin with human triple negative breast cancer cells(MDA-MB-231). FIG. 26A is a series of images at 10× magnification ofplates demonstrating attenuation of migration into the wound in OS2966and composite human variants (H1, H2, H3) treated wells. FIG. 26B is agraph of quantitation of cell free area for each condition (performed intriplicate and repeated).

FIGS. 27A-B is a series of pictorial and graphical representationsillustrating functional validation in tube forming angiogenesis assayswith human umbilical vein endothelial cells (HUVEC) of composite humanantibody variants of the present invention. Functional inhibition of theintegrin β1 subunit with composite human antibody variants of thepresent invention (from Table 2) was assessed in an in vitro model ofangiogenesis; the tube forming assay with human umbilical veinendothelial cells (HUVEC). FIG. 27A is a series of images at 10×magnification of plates demonstrating attenuation of vascular tubeformation in OS2966 and composite human variant (H1, H2, H3) treatedwells. FIG. 27B is a series of graphs of quantitation of closed unitformation for each condition (performed in at least triplicate andrepeated with endothelial progenitor cells).

FIGS. 28A-B is a series of pictorial and graphical representationsillustrating functional validation in a human orthotopic xenograft modelof triple negative breast cancer of composite human antibody variants ofthe present invention. Functional inhibition of the integrin β1 subunitwith composite human antibody variants of the present invention (fromTable 2) was assessed in an in vivo model of human triple negativebreast cancer with the MDA-MB-231 cell line. FIG. 28A is a graph showinggroup tumor mean volume per time. FIG. 28B is an image of a westernblot.

FIG. 29 is a series of pictorial representations illustrating functionalvalidation in a human orthotopic xenograft model of spontaneous lungmetastasis from triple negative breast cancer of composite humanantibody variants of the present invention.

FIGS. 30A-C is a series of pictorial and graphical representationsillustrating functional validation in a human xenograft model ofpancreatic cancer of composite human antibody variants of the presentinvention. Functional inhibition of the integrin β1 subunit withcomposite human antibody variants of the present invention (from Table2) was assessed in an in vivo model of human gemcitabine resistantpancreatic cancer with the PANC1-GEMR cell line. FIG. 30A is a graphshowing group tumor mean volume per time. FIG. 30B is a graph showinggroup tumor mean volume per time. FIG. 30C is an image of a westernblot.

FIGS. 31A-B is a series of pictorial and graphical representationsillustrating functional validation in a human xenograft model ofestablished glioblasoma of composite human antibody variants of thepresent invention. Functional inhibition of the integrin β1 subunit withcomposite human antibody variants of the present invention (from Table2) was assessed in an in vivo model of established human glioblastomawith the U87MG cell line. FIG. 31A is a graph showing group tumor meanvolume per time. FIG. 3B is an image of a blot.

FIGS. 32A-B depicts results of an immunogenicity screening assay(EpiScreen™). FIG. 32A is a histogram. FIG. 32B is a summary of healthydonor T cell proliferation responses to the donor cohort.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides human VH and VL framework sequences andnucleic acid sequences that encode them. Such sequences are used, forexample, to provide frameworks for grafting CDRs from a donor antibody,e.g., a rodent antibody. Thus, an antibody comprising a VH and/or VLframework of the invention with the binding specificity of a donorantibody can be created.

The invention also provides humanized antibodies having the specificityof an antibody termed OS2966, e.g., specific binding to integrin β1, viaCDRs of murine OS2966 provided in the human VH and VL framework regionsset forth in SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 29-57, 91-100and 107-116.

The humanized antibodies of the invention are used for a variety oftherapeutic and diagnostic purposes as described herein. Uses includediagnosing and treating diseases such as cancer.

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to the particular device,methods, and experimental conditions described, as such devices,methods, and conditions may vary. It is also to be understood that theterminology used herein is for purposes of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only in the appended claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to “thecomposition” or “the method” includes one or more compositions andmethods, and/or steps of the type described herein which will becomeapparent to those persons skilled in the art upon reading thisdisclosure and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, the preferred methods andmaterials are now described.

The term “integrin β1” is synonymous with CD29 and includes reference toa protein encoded by the ITGB1 gene. Integrin β1 is associated with lateantigen receptors and is known to conjoin with a number of alphasubunits including alpha-1 though alpha-9, for example, complexing toalpha-3 subunit creates α3β1 complex that reacts to such molecules asnetrin-1 and reelin.

As used herein, the term “anti-integrin β1” in reference to an antibody,refers to an antibody that specifically binds integrin β1.

The term “antibody” refers to a polypeptide encoded by an immunoglobulingene or functional fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain (VL)and variable heavy chain (VH) refer to these light and heavy chainsrespectively.

Examples of antibody functional fragments include, but are not limitedto, complete antibody molecules, antibody fragments, such as Fv, singlechain Fv (scFv), complementarity determining regions (CDRs), VL (lightchain variable region), VH (heavy chain variable region), Fab, F(ab)2′and any combination of those or any other functional portion of animmunoglobulin peptide capable of binding to target antigen (see, e.g.,Fundamental Immunology (Paul ed., 3d ed. 1993). As appreciated by one ofskill in the art, various antibody fragments can be obtained by avariety of methods, for example, digestion of an intact antibody with anenzyme, such as pepsin; or de novo synthesis. Antibody fragments areoften synthesized de novo either chemically or by using recombinant DNAmethodology. Thus, the term antibody, as used herein, includes antibodyfragments either produced by the modification of whole antibodies, orthose synthesized de novo using recombinant DNA methodologies (e.g.,single chain Fv) or those identified using phage display libraries. Theterm antibody also includes bivalent or bispecific molecules, diabodies,triabodies, and tetrabodies. Bivalent and bispecific molecules are knownin the art.

References to “VH” or a “VH” refer to the variable region of animmunoglobulin heavy chain, including an Fv, scFv, adisulfilde-stabilized Fv (dsFv) or Fab. References to “VL” or a “VL”refer to the variable region of an immunoglobulin light chain, includingof an Fv, scFv, dsFv or Fab.

The CDRs are primarily responsible for binding to an epitope of anantigen. The CDRs of each chain are typically referred to as CDR1, CDR2,and CDR3, numbered sequentially starting from the N-terminus, and arealso typically identified by the chain in which the particular CDR islocated. Thus, a VH CDR3 is located in the variable domain of the heavychain of the antibody in which it is found, whereas a VL CDR1 is theCDR1 from the variable domain of the light chain of the antibody inwhich it is found. The numbering of the light and heavy chain variableregions described herein is in accordance with Kabat (see, e.g., Johnsonet al., (2001) “Kabat Database and its applications: future directions”Nucleic Acids Research, 29: 205-206; and the Kabat Database of Sequencesof Proteins of Immunological Interest, Feb. 22, 2002 Dataset) unlessotherwise indicated.

The positions of the CDRs and framework regions can be determined usingvarious well known definitions in the art, e.g., Kabat, Chothia,international ImMunoGeneTics database (IMGT), and AbM (see, e.g.,Johnson et al., supra; Chothia & Lesk, 1987, Canonical structures forthe hypervariable regions of immunoglobulins. J. Mol. Biol. 196,901-917; Chothia C. et al., 1989, Conformations of immunoglobulinhypervariable regions. Nature 342, 877-883; Chothia C. et al., 1992,structural repertoire of the human VH segments J. Mol. Biol. 227,799-817; Al-Lazikani et al., J. Mol. Biol. 1997, 273(4)). Definitions ofantigen combining sites are also described in the following: Ruiz etal., IMGT, the international ImMunoGeneTics database. Nucleic AcidsRes., 28, 219-221 (2000); and Lefranc, M.-P. IMGT, the internationalImMunoGeneTics database. Nucleic Acids Res. January 1; 29(1):207-9(2001); MacCallum et al, Antibody-antigen interactions: Contact analysisand binding site topography, J. Mol. Biol., 262 (5), 732-745 (1996); andMartin et al, Proc. Natl. Acad. Sci. USA, 86, 9268-9272 (1989); Martin,et al, Methods Enzymol., 203, 121-153, (1991); Pedersen et al,Immunomethods, 1, 126, (1992); and Rees et al, In Sternberg M. J. E.(ed.), Protein Structure Prediction. Oxford University Press, Oxford,141-172 1996).

Exemplary framework and CDR sequences for human VH and VL regionsdisclosed herein are shown in FIG. 4.

“OS2966” refers to a murine IgG1 antibody that specifically binds tohuman integrin β1. OS2966 is commercially available (under a differentdesignation) from several sources, such as the Developmental StudiesHybridoma Bank of the University of Iowa. The heavy and light chains ofOS2966 have been cloned. The nucleotide and amino acid sequences of theOS2966 VH region are set forth in SEQ ID NO:1 and SEQ ID NO:2,respectively. The nucleotide and amino acid sequences of the OS2966 VLregion are set forth in SEQ ID NO:3 and SEQ ID NO:4, respectively. TheOS2966 CDRs as designated for the exemplary humanized antibodiesdescribed herein are set forth in SEQ ID NOs:23-28 and shown in FIG. 4.

A “humanized antibody” refers to an antibody that comprises a donorantibody binding specificity, i.e., the CDR regions of a donor antibody,typically a mouse monoclonal antibody, grafted onto human frameworksequences. A “humanized antibody” as used herein binds to the sameepitope as the donor antibody and typically has at least 25% of thebinding affinity. An exemplary assay for binding affinity is describedin Example 5. Methods to determine whether the antibody binds to thesame epitope are well known in the art, see, e.g., Harlow & Lane, UsingAntibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press,1999, which discloses techniques to epitope mapping or alternatively,competition experiments, to determine whether an antibody binds to thesame epitope as the donor antibody. A humanized antibody that comprisesa novel framework region provided in the invention.

A “VH” or “VL” “region” or “framework” of the invention refers to the aVH or VL amino acid sequence that has at least 70% identity, often, atleast 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity, to anamino acid sequence set forth in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10,SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20,SEQ ID NO:22, SEQ ID NOs:29-57, SEQ ID NOs:91-100, or SEQ IDNOs:107-116. A “framework” of a VH or VL chain refers to the frameworkregions of the chain not including the CDRs. The term as applied to eachchain encompasses all of the framework regions.

A “humanized anti-integrin β1 antibody” refers to a humanized antibodycomprising a human framework sequence that has the binding specificityof the murine OS2966 grafted to that framework. A CDR of a humanizedanti-integrin β1 antibody of the invention has at least 85%, moretypically at least 90%, 95%, 96%, 97%, 98%, or 99% identity to a CDR ofthe heavy and light chain sequences set forth in SEQ ID NO:2 and SEQ IDNO:4, respectively. CDRs of the VH region are set forth in SEQ ID NO:23,SEQ ID NO:24, and SEQ ID NO:25. CDRs of the VH region are set forth inSEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28.

In one aspect, the invention provides composite humanized antibodies.Composite human antibody technology generates humanized non-immunogenicantibodies by avoiding T cell epitopes (deimmunisation) in variableregion (V region) sequences (EP2,388,871). Unlike other humanizationtechnologies that use single human V region frameworks as ‘acceptors’for complimentarity-determining regions (CDRs) from the startingantibody (typically murine), Composite Human Antibodies™ comprisemultiple sequence segments (composites') derived from V regions ofunrelated human antibodies. The key properties of Composite HumanAntibodies are as follows:

Sequence segments derived from databases of unrelated human V regionsare selected after determining amino acids which are considered criticalfor antigen binding of the starting antibody. All selected sequencesegments derived from human V region databases are filtered for thepresence of potential T cell epitopes using Antitope's in silico tools.Composite Human Antibodies™ retain affinity and specificity better thanstandard humanized antibodies due to the close fit of human sequencesegments with all sections of the starting antibody V regions. CompositeHuman Antibodies™ are depleted of T cell epitopes and thereforeconsidered both humanized and deimmunised.

In one embodiment, the invention antibodies are prepared by identifyingcandidate residues in the framework region to be mutated at specificsites within T cell epitopes. Invention antibodies may exhibit alteredbinding affinity and/or altered immunogenicity as compared to donorantibodies.

Methods known in the art can be used to map T cell epitopes within aprotein sequence. For example, EpiScreen™ (EP1989544, Antitope, UK) isused to map T cell epitopes within a protein sequence to determinepotential for immunogenicity, which is based on the number and potencyof T cell epitopes within a sequence. EpiScreen™ T cell epitope mappingtypically uses CD8+ T cell depleted PBMCs from a minimum of 50 HLA-typeddonors (selected to represent the human population of interest).Typically, 15mer peptides with 12 amino acid overlaps spanning a proteinsequence are analyzed in a large number of replicate cultures for invitro CD4+ T cell stimulation by 3H TdR incorporation. CD4+ T cellstimulation is often detected in two or three adjacent and overlappingpeptides since the core 9mer that binds the MHC class II binding groovewill be present in more than one peptide sequence. After the accurateidentification of peptides that stimulate CD4+ T cells in vitro, insilico technologies can be used to design epitope-depleted (deimmunized)variants by determining the precise location of core 9mer sequences andthe location of key MHC class II anchor residues.

The phrase “single chain Fv” or “scFv” refers to an antibody in whichthe variable domains of the heavy chain and of the light chain of atraditional two chain antibody have been joined to form one chain.Typically, a linker peptide is inserted between the two chains to allowfor the stabilization of the variable domains without interfering withthe proper folding and creation of an active binding site. A singlechain humanized antibody of the invention, e.g., humanized anti-integrinβ1 antibody, may bind as a monomer. Other exemplary single chainantibodies may form diabodies, triabodies, and tetrabodies. (See, e.g.,Hollinger et al., 1993, supra). Further the humanized antibodies of theinvention, e.g., humanized anti-integrin β1 antibody may also form onecomponent of a “reconstituted” antibody or antibody fragment, e.g., aFab, a Fab′ monomer, a F(ab)′2 dimer, or an whole immunoglobulinmolecule. Thus, a humanized antibody of the present invention mayfurther comprise a human Fc region.

“Join” or “link” or “conjugate” refers to any method known in the artfor functionally connecting protein domains, including withoutlimitation recombinant fusion with or without intervening domains,intein-mediated fusion, non-covalent association, and covalent bonding,e.g., disulfide bonding, peptide bonding; hydrogen bonding;electrostatic bonding; and conformational bonding, e.g.,antibody-antigen, and biotin-avidin associations. In the context of thepresent invention, the terms include reference to joining an antibodymoiety to an effector molecule (EM). The linkage can be either bychemical or recombinant means. Chemical means refers to a reactionbetween the antibody moiety and the effector molecule such that there isa covalent bond formed between the two molecules to form one molecule.

The term “effector moiety” means the portion of an immunoconjugateintended to have an effect on a cell targeted by the targeting moiety orto identify the presence of the immunoconjugate. Thus, the effectormoiety can be, for example, a therapeutic moiety, such as a cytotoxicagent or drug, or a detectable moiety, such as a fluorescent label.

A “therapeutic moiety” is the portion of an immunoconjugate intended toact as a therapeutic agent.

The term “therapeutic agent” includes any number of compounds currentlyknown or later developed to act as chemotherapeutic agents,anti-neoplastic compounds, anti-inflammatory compounds, anti-infectivecompounds, enzyme activators or inhibitors, allosteric modifiers,antibiotics or other agents administered to induce a desired therapeuticeffect in a patient. The therapeutic agent may also be a toxin or aradioisotope, where the therapeutic effect intended is, for example, thekilling of a cancer cell.

The terms “effective amount” or “amount effective to” or“therapeutically effective amount” refers to an amount sufficient toinduce a detectable therapeutic response in the subject. Preferably, thetherapeutic response is effective in reducing the proliferation ofcancer cells or in inhibiting the growth of cancer cells present in asubject. Assays for determining therapeutic responses are well known inthe art.

The term “immunoconjugate” refers to a composition comprising anantibody linked to a second molecule such as a detectable label oreffector molecule. Often, the antibody is linked to the second moleculeby covalent linkage.

In the context of an immunoconjugate, a “detectable label” or“detectable moiety” refers to, a portion of the immunoconjugate whichhas a property rendering its presence detectable. For example, theimmunoconjugate may be labeled with a radioactive isotope which permitscells in which the immunoconjugate is present to be detected inimmunohistochemical assays. A “detectable label” or a “detectablemoiety” is a composition detectable by spectroscopic, photochemical,biochemical, immunochemical, chemical, or other physical means. Forexample, useful labels include radioisotopes (e.g., ³H, ³⁵S, ³²P, ⁵¹Cr,or ¹²⁵I), fluorescent dyes, electron-dense reagents, enzymes (e.g.,alkaline phosphatase, horseradish peroxidase, or others commonly used inan ELISA), biotin, digoxigenin, or haptens and proteins which can bemade detectable, e.g., by incorporating a radiolabel into the peptide orused to detect antibodies specifically reactive with the peptide.

The term “immunologically reactive conditions” includes reference toconditions which allow an antibody generated to a particular epitope tobind to that epitope to a detectably greater degree than, and/or to thesubstantial exclusion of, binding to substantially all other epitopes.Immunologically reactive conditions are dependent upon the format of theantibody binding reaction and typically are those utilized inimmunoassay protocols or those conditions encountered in vivo. SeeHarlow & Lane, supra, for a description of immunoassay formats andconditions. Preferably, the immunologically reactive conditions employedin the methods of the present invention are “physiological conditions”which include reference to conditions (e.g., temperature, osmolarity,pH) that are typical inside a living mammal or a mammalian cell. Whileit is recognized that some organs are subject to extreme conditions, theintra-organismal and intracellular environment normally lies around pH 7(i.e., from pH 6.0 to pH 8.0, more typically pH 6.5 to 7.5), containswater as the predominant solvent, and exists at a temperature above0.degree. C. and below 50.degree. C. Osmolarity is within the range thatis supportive of cell viability and proliferation.

The term “specifically binds,” “binding specificity,” “specificallybinds to an antibody” or “specifically immunoreactive with,” whenreferring to an epitope, refers to a binding reaction which isdeterminative of the presence of the epitope in a heterogeneouspopulation of proteins and other biologics. Thus, under designatedimmunoassay conditions, the specified antibodies bind to a particularepitope at least two times the background and more typically more than10 to 100 times background. A variety of immunoassay formats may be usedto select antibodies specifically immunoreactive with a particularprotein or carbohydrate. For example, solid-phase ELISA immunoassays areroutinely used to select antibodies specifically immunoreactive with aprotein or carbohydrate. See, Harlow & Lane, ANTIBODIES, A LABORATORYMANUAL, Cold Spring Harbor Press, New York (1988) and Harlow & Lane,USING ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Press, NewYork (1999), for a description of immunoassay formats and conditionsthat can be used to determine specific immunoreactivity. As used herein,“specifically binds” means that an antibody binds to a protein with a Kdof at least about 0.1 mM, at least about 1 μM, at least about 0.1 μM orbetter, or 0.01 μM or better.

“Nucleic acid” and “polynucleotide” are used interchangeably herein torefer to deoxyribonucleotides or ribonucleotides and polymers thereof ineither single- or double-stranded form. The term encompasses nucleicacids containing known nucleotide analogs or modified backbone residuesor linkages, which are synthetic, naturally occurring, and non-naturallyoccurring, which have similar binding properties as the referencenucleic acid, and which are metabolized in a manner similar to thereference nucleotides. Examples of such analogs include, withoutlimitation, phosphorothioates, phosphoramidates, methyl phosphonates,chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleicacids (PNAs). As appreciate by one of skill in the art, the complementof a nucleic acid sequence can readily be determined from the sequenceof the other strand. Thus, any particular nucleic acid sequence setforth herein also discloses the complementary strand.

“Polypeptide,” “peptide” and “protein” are used interchangeably hereinto refer to a polymer of amino acid residues. The terms apply tonaturally occurring amino acid polymers, as well as, amino acid polymersin which one or more amino acid residue is an artificial chemicalmimetic of a corresponding naturally occurring amino acid.

Amino acid” refers to naturally occurring and synthetic amino acids, aswell as amino acid analogs and amino acid mimetics that function in amanner similar to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,.gamma.-carboxyglutamate, and O-phosphoserine. “Amino acid analogs”refers to compounds that have the same fundamental chemical structure asa naturally occurring amino acid, i.e., an alpha carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. “Amino acid mimetics” refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid. Amino acids may bereferred to herein by either their commonly known three letter symbolsor by the one-letter symbols recommended by the IUPAC-IUB BiochemicalNomenclature Commission.

“Conservatively modified variants” applies to both nucleic acid andamino acid sequences. With respect to particular nucleic acid sequences,conservatively modified variants refers to those nucleic acids whichencode identical or essentially identical amino acid sequences, or wherethe nucleic acid does not encode an amino acid sequence, to essentiallyidentical sequences. Because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode any givenprotein. For instance, the codons GCA, GCC, GCG and GCU all encode theamino acid alanine. Thus, at every position where an alanine isspecified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations,” which are onespecies of conservatively modified variations. Every nucleic acidsequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

With respect to amino acid sequences, one of skill will recognize thatindividual substitutions, deletions or additions to a nucleic acid,peptide, polypeptide, or protein sequence which alters, adds or deletesa single amino acid or a small percentage of amino acids in the encodedsequence is a “conservatively modified variant” where the alterationresults in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. Such conservativelymodified variants are in addition to and do not exclude polymorphicvariants, interspecies homologues, and alleles of the invention.

For example, substitutions may be made wherein an aliphatic amino acid(G, A, I, L, or V) is substituted with another member of the group, orsubstitution such as the substitution of one polar residue for another,such as arginine for lysine, glutamic for aspartic acid, or glutaminefor asparagine. Each of the following eight groups contains otherexemplary amino acids that are conservative substitutions for oneanother: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamicacid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g.,Creighton, Proteins (1984)).

Macromolecular structures such as polypeptide structures can bedescribed in terms of various levels of organization. For a generaldiscussion of this organization, see, e.g., Alberts et al., MolecularBiology of the Cell (3rd ed., 1994) and Cantor and Schimmel, BiophysicalChemistry Part I. The Conformation of Biological Macromolecules (1980).“Primary structure” refers to the amino acid sequence of a particularpeptide. “Secondary structure” refers to locally ordered, threedimensional structures within a polypeptide. “Tertiary structure” refersto the complete three dimensional structure of a polypeptide monomer.Domains are portions of a polypeptide that form a compact unit of thepolypeptide and are typically 50 to 350 amino acids long. Typicaldomains are made up of sections of lesser organization such as stretchesof β-sheet and α-helices. Quaternary structure” refers to the threedimensional structure formed by the noncovalent association ofindependent tertiary units.

The terms “isolated” or “substantially purified,” when applied to anucleic acid or protein, denotes that the nucleic acid or protein isessentially free of other cellular components with which it isassociated in the natural state. It is preferably in a homogeneousstate, although it can be in either a dry or aqueous solution. Purityand homogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein which is the predominantspecies present in a preparation is substantially purified.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over aspecified region, when compared and aligned for maximum correspondenceover a comparison window or designated region) as measured using a BLASTor BLAST 2.0 sequence comparison algorithms with default parametersdescribed below, or by manual alignment and visual inspection. Suchsequences are then said to be “substantially identical.” This definitionalso refers to, or may be applied to, the compliment of a test sequence.The definition also includes sequences that have deletions and/oradditions, as well as those that have substitutions. As described below,the preferred algorithms can account for gaps and the like. Preferably,identity exists over a region that is at least about 25 amino acids ornucleotides in length, or more preferably over a region that is 50-100amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local alignment algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the globalalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).The Smith & Waterman alignment with the default parameters are oftenused when comparing sequences as described herein.

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403410(1990), respectively. BLAST and BLAST 2.0 are used, typically with thedefault parameters, to determine percent sequence identity for thenucleic acids and proteins of the invention. Software for performingBLAST analyses is publicly available through the National Center forBiotechnology Information. This algorithm involves first identifyinghigh scoring sequence pairs (HSPs) by identifying short words of lengthW in the query sequence, which either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighborhood wordscore threshold. These initial neighborhood word hits act as seeds forinitiating searches to find longer HSPs containing them. The word hitsare extended in both directions along each sequence for as far as thecumulative alignment score can be increased. Cumulative scores arecalculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always >0) and N (penalty scorefor mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and acomparison of both strands. For amino acid (protein) sequences, theBLASTP program uses as defaults a wordlength (W) of 3, an expectation(E) of 10, and the BLOSUM62 scoring matrix (see Henikoff& Henikoff(1989) Proc. Natl. Acad. Sci. USA 89:10915)). For the purposes of thisinvention, the BLAST2.0 algorithm is used with the default parameters.

A “phage display library” refers to a “library” of bacteriophages onwhose surface is expressed exogenous peptides or proteins. The foreignpeptides or polypeptides are displayed on the phage capsid outersurface. The foreign peptide can be displayed as recombinant fusionproteins incorporated as part of a phage coat protein, as recombinantfusion proteins that are not normally phage coat proteins, but which areable to become incorporated into the capsid outer surface, or asproteins or peptides that become linked, covalently or not, to suchproteins. This is accomplished by inserting an exogenous nucleic acidsequence into a nucleic acid that can be packaged into phage particles.Such exogenous nucleic acid sequences may be inserted, for example, intothe coding sequence of a phage coat protein gene. If the foreignsequence is cloned in frame, the protein it encodes will be expressed aspart of the coat protein. Thus, libraries of nucleic acid sequences,such as that of an antibody repertoires made from the gene segmentsencoding the entire B cell repertoire of one or more individuals, can beso inserted into phages to create “phage libraries.” As peptides andproteins representative of those encoded for by the nucleic acid libraryare displayed by the phage, a “peptide-display library” is generated.While a variety of bacteriophages are used in such libraryconstructions, typically, filamentous phage are used (Dunn (1996) Curr.Opin. Biotechnol. 7:547-553). See, e.g., description of phage displaylibraries, below.

The term “chimeric antibodies” refers to antibodies wherein the aminoacid sequence of the immunoglobulin molecule is derived from two or morespecies. Typically, the variable region of both light and heavy chainscorresponds to the variable region of antibodies derived from onespecies of mammals (e.g. mouse, rat, rabbit, and the like) with thedesired specificity, affinity, and capability while the constant regionsare homologous to the sequences in antibodies derived from another(usually human) to avoid eliciting an immune response in that species.

The term “epitope” or “antigenic determinant” or “antigen determinationregion” are used interchangeably herein and refer to that portion of anantigen capable of being recognized and specifically bound by aparticular antibody. When the antigen is a polypeptide, epitopes can beformed both from contiguous amino acids and noncontiguous amino acidsjuxtaposed by tertiary folding of a protein. Epitopes formed fromcontiguous amino acids are typically retained upon protein denaturing,whereas epitopes formed by tertiary folding are typically lost uponprotein denaturing. An epitope typically includes at least 3, and moreusually, at least 5 or 8-10 amino acids in a unique spatialconformation. An antigenic determinant can compete with the intactantigen (i.e., the “immunogen” used to elicit the immune response) forbinding to an antibody.

Competition between antibodies is determined by an assay in which theimmunoglobulin under test inhibits specific binding of a referenceantibody to a common antigen. Numerous types of competitive bindingassays are known, for example: solid phase direct or indirectradioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay (see Stahli et al.,Methods in Enzymology 9:242-253 (1983)); solid phase directbiotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614-3619(1986)); solid phase direct labeled assay, solid phase direct labeledsandwich assay (see Harlow and Lane, “Antibodies, A Laboratory Manual,”Cold Spring Harbor Press (1988)); solid phase direct label RIA using1-125 label (see Morel et al., Molec. Immunol. 25(1):7-15 (1988)); solidphase direct biotin-avidin EIA (Cheung et al., Virology 176:546-552(1990)); and direct labeled RIA (Moldenhauer et al., Scand. J. Immunol.32:77-82 (1990)). Typically, such an assay involves the use of purifiedantigen bound to a solid surface or cells bearing either of these, anunlabeled test immunoglobulin and a labeled reference immunoglobulin.Competitive inhibition is measured by determining the amount of labelbound to the solid surface or cells in the presence of the testimmunoglobulin. Usually the test immunoglobulin is present in excess.Antibodies identified by competition assay (competing antibodies)include antibodies binding to the same epitope as the reference antibodyand antibodies binding to an adjacent epitope sufficiently proximal tothe epitope bound by the reference antibody for steric hindrance tooccur. Usually, when a competing antibody is present in excess, it willinhibit specific binding of a reference antibody to a common antigen byat least 50 or 75%.

Antibodies of the present invention, e.g, VH polypeptides, VLpolypeptides, or single chain antibodies, may be generated using routinetechniques in the field of recombinant genetics. Basic texts disclosingthe general methods used in this invention include Sambrook & Russell,Molecular Cloning, A Laboratory Manual (3d ed. 2001) and CurrentProtocols in Molecular Biology (Ausubel et al., eds., 1999).

Humanized antibodies of the invention may be generated by grafting thespecificity, i.e., the antigen binding loops, of a donor antibody,typically a murine antibody, to a human framework. The framework regionsof the human light chain and heavy chains provided herein can readily bedetermined by the practitioner. The position numbers of the heavy andlight chains are designated in accordance with common numbering schemes,e.g., the Kabat and Chothia numbering scheme. The Chothia number schemeis identical to the Kabat scheme, but places the insertions in CDR-L1and CDR-H1 at structurally different positions. Unless otherwiseindicated, the Kabat numbering scheme is used herein in reference to thesequence positions. The position of an amino acid residue in aparticular VH or VL sequence does not refer to the number of amino acidsin a particular sequence, but rather refers to the position asdesignated with reference to a numbering scheme.

The positions of the CDRs and hence the positions of the frameworkregions of the human heavy chain and light chains are determined usingdefinitions that are standard in the field. For example, the followingfour definitions are commonly used. The Kabat definition is based onsequence variability and is the most commonly used. The Chothiadefinition is based on the location of the structural loop regions. TheAbM definition is a compromise between the two used by OxfordMolecular's AbM antibody modelling software. The contact definition hasbeen recently introduced and is based on an analysis of the availablecomplex crystal structures. The following are the loop positions, i.e.,CDRs, using the four different definitions.

A VH or VL sequence of the invention comprises a heavy or light chainthat typically has at least 70% identity, more typically 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identity to a sequence comprised by SEQID NOs:5-22, 29-57, 91-100 or 107-116.

Furthermore, a number of important residues have been identified outsideof the CDRs of OS2966 which are preferably utilized in the humanized VHand VL chains. For example, in one embodiment, one or more amino acidresidues in the VH chain are identical to that of OS2966 (SEQ ID NO:2),including residues 48, 67, 69, 73, 76, 80, 89, 91 and 93. In oneembodiment, one or more amino acid residues in the VL chain areidentical to that of OS2966 (SEQ ID NO:4), including residues 36 and 71.

A humanized antibody of the invention binds to the same epitope as thedonor antibody, e.g., binds to the same integrin β1 epitope, or competesfor binding to the same integrin β1 epitope, that OS2966 binds to, forexample. Methods to determine whether the antibody binds to the sameepitope are well known in the art, see, e.g., Harlow & Lane, UsingAntibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press,1999, which discloses techniques to epitope mapping or alternatively,competition experiments, to determine whether an antibody binds to thesame epitope as the donor antibody.

A stable humanized antibody of the invention may exhibit alteredaffinity when compared to the donor antibody. For example, in someembodiments, the affinity of a single chain humanized anti-integrin β1,may, for example, be decreased compared to a single chain antibodycomprising the OS2966 VH and VL regions. Such a decrease may be by asmuch as 10-fold in comparison, but typically a humanized antibody of theinvention has an affinity that is at least 25%, more often at least 50%of that of the comparable wildtype antibody. (A “comparable wildtypeantibody” refers to an antibody of the same embodiment, e.g., scFv, thatcomprises the donor antibody VH and VL regions). In some embodiments,the affinity for the epitope is increased, such that a humanizedantibody of the invention has an affinity that is 2 times and sometime5, 10, 50, or 100 times the affinity of the comparable wildtypeantibody.

The heavy and light chain regions of the invention are typicallyobtained using recombinant DNA technology. The recombinant DNAmethodologies that are commonly employed to perform this are well knownto those of skill in the art. Typically, nucleic acid sequences encodingthe frameworks and CDRs of the donor antibodies are generated by PCR,for example by overlap extension. In this technique, the antigen bindingsequences of the donor antibody are typically joined to the humanframework regions by incorporating the desired sequences intooligonucleotides and creating a series of products using PCR thatcomprise the desired donor and human sequences. The products may then bejoined, typically using additional PCR reactions, in the properorientation to create the VH and VL chains that comprise human frameworkregions with donor antibody CDRs. The VL and VH DNA sequences may beligated together, either directly or through a DNA sequence encoding apeptide linker, using techniques well known to those of skill in theart. These techniques include PCR as well as techniques such as in vitroligation. The VL and VH sequences may be linked in either orientation.

Examples of techniques sufficient to direct persons of skill through invitro amplification methods are well known in the art.

Oligonucleotides that are not commercially available can be chemicallysynthesized according to the solid phase phosphoramidite triester methodfirst described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862(1981), using an automated synthesizer, as described in Van Devanter et.al., Nucleic Acids Res. 12:6159-6168 (1984). Purification ofoligonucleotides is by either native acrylamide gel electrophoresis orby anion-exchange HPLC as described in Pearson & Reanier, J. Chrom.255:137-149 (1983).

The sequence of the cloned genes and synthetic oligonucleotides can beverified after cloning using, e.g., by sequencing.

PCR products are subcloned into suitable cloning vectors that are wellknown to those of skill in the art and commercially available. Thenucleotide sequence of the heavy or light chain coding regions is thendetermined.

One of skill will appreciate that, utilizing the sequence informationprovided for the variable regions, nucleic acids encoding thesesequences are obtained using any number of additional methods well knownto those of skill in the art. Thus, DNA encoding the Fv regions isprepared by any suitable method, including, for example, otheramplification techniques such as ligase chain reaction (LCR),transcription amplification, and self-sustained sequence replication, orcloning and restriction of appropriate sequences.

The nucleic acids encoding the antibodies and antibody fragments of theinvention can also be generated by direct chemical synthesis usingmethods such as the phosphotriester method; the phosphodiester method;the diethylphosphoramidite method; and the solid support method of U.S.Pat. No. 4,458,066. If the DNA sequence is synthesized chemically, asingle stranded oligonucleotide will result. This may be converted intodouble stranded DNA by hybridization with a complementary sequence, orby polymerization with a DNA polymerase using the single strand as atemplate. While it is possible to chemically synthesize an entire singlechain Fv region, it is preferable to synthesize a number of shortersequences (about 100 to 150 bases) that are typically later splicedtogether, for example using overlap extension PCR.

For nucleic acids, sizes are given in either kilobases (kb) or basepairs (bp). These are estimates derived from agarose or acrylamide gelelectrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Protein sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

The VH and VL domains of an antibody of the invention may be directlylinked or may be separated by a linker, e.g. to stabilize the variableantibody domains of the light chain and heavy chain, respectively.Suitable linkers are well known to those of skill in the art and includethe well known GlyGlyGlyGlySer (SEQ ID NO:122) linker or a variantthereof. For example, a typical linker is (Gly4Ser)₃ (SEQ ID NO:123).Other linkers, including hinge regions, that can be used in theinvention include those described, for example in Alfthan et al, ProteinEng. 8(7), 725-31; Choi et al, Eur. J. Immunol. 31(1), 94-106; Hu et al,Cancer Res. 56(13), 3055-61; Kipriyanov, et al, Protein Eng. 10(4),445-53; Pack, et al, Biotechnology (N Y) 11(11), 1271-7; and Roovers, etal, Cancer Immunol. Immunother. 50(1):51-9.

To obtain high level expression of a cloned gene or nucleic acid, suchas those cDNAs encoding the humanized antibodies, e.g., a humanizedantibody of the invention, or an immunoconjugate or chimeric antibodycomprising a humanized antibody of the invention, one typicallysubclones a nucleic acid encoding the antibody or immunoconjugate intoan expression vector that contains an appropriate promoter to directtranscription, a transcription/translation terminator, and if for anucleic acid encoding a protein, a ribosome binding site fortranslational initiation. Suitable bacterial promoters are well known inthe art and described, e.g., in Sambrook et al. and Ausubel et al.Bacterial expression systems for expressing protein are available in,e.g., E. coli, Bacillus sp., and Salmonella (Palva et al., Gene22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983). Kits forsuch expression systems are commercially available. Eukaryoticexpression systems for mammalian cells, yeast, and insect cells are wellknown in the art and are also commercially available.

Often, in order to express a protein at high levels in a cell, codonpreference for the expression system is considered in constructing thenucleic acid sequence to be expressed. Thus, a nucleic acid from oneorganism, e.g., a human or mouse, may be engineered to accommodate thecodon preference of the expression system.

The promoter used to direct expression of a heterologous nucleic aciddepends on the particular application. The promoter is optionallypositioned about the same distance from the heterologous transcriptionstart site as it is from the transcription start site in its naturalsetting. As is known in the art, however, some variation in thisdistance can be accommodated without loss of promoter function.

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the protein-encodingnucleic acid in host cells. A typical expression cassette thus containsa promoter operably linked to the nucleic acid sequence encoding theprotein to be expressed and signals required for efficientpolyadenylation of the transcript, ribosome binding sites, andtranslation termination. The nucleic acid sequence encoding a proteinmay typically be linked to a cleavable signal peptide sequence topromote secretion of the encoded protein by the transformed cell. Suchsignal peptides would include, among others, the signal peptides fromtissue plasminogen activator, insulin, and neuron growth factor, andjuvenile hormone esterase of Heliothis virescens. Additional elements ofthe cassette may include enhancers and, if genomic DNA is used as thestructural gene, introns with functional splice donor and acceptorsites.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

Expression control sequences that are suitable for use in a particularhost cell are often obtained by cloning a gene that is expressed in thatcell. Commonly used prokaryotic control sequences, which are definedherein to include promoters for transcription initiation, optionallywith an operator, along with ribosome binding site sequences, includesuch commonly used promoters as the beta-lactamase (penicillinase) andlactose (lac) promoter systems (Change et al., Nature (1977) 198: 1056),the tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res.(1980) 8: 4057), the tac promoter (DeBoer, et al., Proc. Natl. Acad.Sci. U.S.A. (1983) 80:21-25); and the lambda-derived P.sub.L promoterand N-gene ribosome binding site (Shimatake et al., Nature (1981) 292:128). The particular promoter system is not critical to the invention,any available promoter that functions in prokaryotes can be used.

Standard bacterial expression vectors include plasmids such aspBR322-based plasmids, e.g., pBLUESCRIPT™, pSKF, pET23D, .lamda.-phagederived vectors, and fusion expression systems such as GST and LacZ.Epitope tags can also be added to recombinant proteins to provideconvenient methods of isolation, e.g., c-myc, HA-tag, 6-His tag (SEQ IDNO:124), maltose binding protein, VSV-G tag, anti-DYKDDDDK tag (SEQ IDNO:125), or any such tag, a large number of which are well known tothose of skill in the art.

Eukaryotic expression systems for mammalian cells, yeast, and insectcells are well known in the art and are also commercially available. Inyeast, vectors include Yeast Integrating plasmids (e.g., YIp5) and YeastReplicating plasmids (the YRp series plasmids) and pGPD-2. Expressionvectors containing regulatory elements from eukaryotic viruses aretypically used in eukaryotic expression vectors, e.g., SV40 vectors,papilloma virus vectors, and vectors derived from Epstein-Barr virus.Other exemplary eukaryotic vectors include pMSG, pAV009/A+, pMTO10/A+,pMAMneo-5, baculovirus pDSVE, and any other vector allowing expressionof proteins under the direction of the CMV promoter, SV40 earlypromoter, SV40 later promoter, metallothionein promoter, murine mammarytumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter,or other promoters shown effective for expression in eukaryotic cells.

Some expression systems have markers that provide gene amplificationsuch as thymidine kinase, hygromycin B phosphotransferase, anddihydrofolate reductase. Alternatively, high yield expression systemsnot involving gene amplification are also suitable, such as using abaculovirus vector in insect cells, with a GPCR-encoding sequence underthe direction of the polyhedrin promoter or other strong baculoviruspromoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of eukaryotic sequences. The particularantibiotic resistance gene chosen is not critical, any of the manyresistance genes known in the art are suitable. The prokaryoticsequences are optionally chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

Standard transfection methods are used to produce bacterial, mammalian,yeast or insect cell lines that express large quantities of protein,which are then purified using standard techniques (see, e.g., Colley etal., J. Biol. Chem. 264:17619-17622 (1989); Guide to ProteinPurification, in Methods in Enzymology, vol. 182 (Deutscher, ed.,1990)). Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques (see, e.g., Morrison, J. Bact.132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology101:347-362 (Wu et al., eds, 1983).

Any of the well known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,liposomes, microinjection, plasma vectors, viral vectors and any of theother well known methods for introducing cloned genomic DNA, cDNA,synthetic DNA or other foreign genetic material into a host cell (see,e.g., Sambrook and Russell, supra). It is only necessary that theparticular genetic engineering procedure used be capable of successfullyintroducing at least one gene into the host cell capable of expressing apolypeptide of the invention.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofthe protein, which is recovered from the culture using standardtechniques identified below.

One of skill would recognize that modifications can be made to a nucleicacid encoding a polypeptide of the present invention (i.e., an antibody,a label or effector, or an immunoconjugate formed using the antibody)without diminishing its biological activity. Some modifications may bemade to facilitate the cloning, expression, or incorporation of thetargeting molecule into a fusion protein. Such modifications are wellknown to those of skill in the art and include, for example, terminationcodons, a methionine added at the amino terminus to provide aninitiation, site, additional amino acids placed on either terminus tocreate conveniently located restriction sites, or additional amino acids(such as poly His) to aid in purification steps.

Once expressed, the recombinant antibodies, immunoconjugates, and/oreffector molecules of the present invention can be purified according tostandard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, and the like(see, generally, R. Scopes, PROTEIN PURIFICATION, Springer-Verlag, N.Y.(1982)). Substantially pure compositions of at least about 90 to 95%homogeneity are preferred, and 98 to 99% or more homogeneity are mostpreferred for pharmaceutical uses. Once purified, partially or tohomogeneity as desired, if to be used therapeutically, the polypeptidesshould be substantially free of endotoxin.

Methods for expression of single chain antibodies and/or refolding to anappropriate active form, including single chain antibodies, frombacteria such as E. coli have been described and are well-known and areapplicable to the antibodies of this invention. See, Buchner, et al.,Anal Biochem. 205:263-270 (1992); Pluckthun, Biotechnology 9:545 (1991);Huse, et al., Science 246:1275 (1989) and Ward, et al., Nature 341:544(1989), all incorporated by reference herein.

Often, functional heterologous proteins from E. coli or other bacteriaare isolated from inclusion bodies and require solubilization usingstrong denaturants, and subsequent refolding. During the solubilizationstep, as is well-known in the art, a reducing agent must be present toseparate disulfide bonds. An exemplary buffer with a reducing agent is:0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol).Reoxidation of the disulfide bonds can occur in the presence of lowmolecular weight thiol reagents in reduced and oxidized form, asdescribed in Saxena, et al., Biochemistry 9: 5015-5021 (1970),incorporated by reference herein, and especially as described byBuchner, et al., supra.

Renaturation is typically accomplished by dilution (e.g., 100-fold) ofthe denatured and reduced protein into refolding buffer. An exemplarybuffer is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM oxidizedglutathione (GSSG), and 2 mM EDTA.

As a modification to the two chain antibody purification protocol, theheavy and light chain regions are separately solubilized and reduced andthen combined in the refolding solution. A preferred yield is obtainedwhen these two proteins are mixed in a molar ratio such that a 5 foldmolar excess of one protein over the other is not exceeded. It isdesirable to add excess oxidized glutathione or other oxidizing lowmolecular weight compounds to the refolding solution after theredox-shuffling is completed.

In addition to recombinant methods, the antibodies and immunoconjugatesof the invention can also be constructed in whole or in part usingstandard peptide synthesis. Solid phase synthesis of the polypeptides ofthe present invention of less than about 50 amino acids in length may beaccomplished by attaching the C-terminal amino acid of the sequence toan insoluble support followed by sequential addition of the remainingamino acids in the sequence. Techniques for solid phase synthesis aredescribed by Barany & Merrifield, THE PEPTIDES: ANALYSIS, SYNTHESIS,BIOLOGY. VOL. 2: SPECIAL METHODS IN PEPTIDE SYNTHESIS, PART A. pp.3-284; Merrifield, et al. J. Am. Chem. Soc. 85:2149-2156 (1963), andStewart, et al., SOLID PHASE PEPTIDE SYNTHESIS, 2ND ED., Pierce Chem.Co., Rockford, Ill. (1984). Proteins of greater length may besynthesized by condensation of the amino and carboxyl termini of shorterfragments. Methods of forming peptide bonds by activation of a carboxylterminal end (e.g., by the use of the coupling reagentN,N′-dicycylohexylcarbodiimide) are known to those of skill.

Conservatively modified variants of antibodies of the present inventionhave at least 80% sequence similarity, often at least 85% sequencesimilarity, 90% sequence similarity, or at least 95%, 96%, 97%, 98%, or99% sequence similarity at the amino acid level, with the protein ofinterest, such as a humanized antibody of the invention.

As noted, the term “conservatively modified variants” can be applied toboth amino acid and nucleic acid sequences. With respect to particularnucleic acid sequences, conservatively modified variants refer to thosenucleic acid sequences which encode identical or essentially identicalamino acid sequences, or if the nucleic acid does not encode an aminoacid sequence, to essentially identical nucleic acid sequences. Becauseof the degeneracy of the genetic code, a large number of functionallyidentical nucleic acids encode any given polypeptide. For instance, thecodons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, atevery position where an alanine is specified by a codon, the codon canbe altered to any of the corresponding codons described without alteringthe encoded polypeptide. Such nucleic acid variations are “silentvariations,” which are one species of conservatively modifiedvariations. Every nucleic acid sequence herein which encodes apolypeptide also describes every possible silent variation of thenucleic acid. One of skill will recognize that each codon in a nucleicacid (except AUG, which is ordinarily the only codon for methionine) canbe modified to yield a functionally identical molecule. Accordingly,each silent variation of a nucleic acid which encodes a polypeptide isimplicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.

One embodiment of the present invention provides an immunoconjugatecomprising a humanized antibody of the invention, linked to an effectormolecule or detectable label. Preferably the effector molecule is atherapeutic molecule such as, for example, a toxin, a chemotherapeuticagent, a small molecule, a cytokine or a chemokine, an enzyme, or aradiolabel. Exemplary toxins include, but are not limited to,Pseudomonas exotoxin or diphtheria toxin. Suitable toxins are describedin e.g., Chaudhary, et al. (1987) Proc Natl Acad Sci USA 84:4538,Chaudhary, et al. (1989) Nature 339:394, Batra, et al. (1991) Mol CellBiol 11:2200. Brinkmann, et al. (1991) Proc Natl Acad Sci USA 88:8616,Siegall, (1995) Semin Cancer Biol 6:289. Examples of small moleculesinclude, but are not limited to, chemotherapeutic compounds such astaxol, doxorubicin, etoposide, and bleiomycin. Exemplary cytokinesinclude, but are not limited to, IL-1, IL-2, IL-4, IL-5, IL-6, andIL-12. Suitable cytokines and chemokines are described in, e.g.,Rosenblum et al. (2000) Int J Cancer 88:267 and Xu et al. (2000) CancerRes 60:4475 and Biragyn et al. (1999) Nat Biotechnol 17:253. Exemplaryenzymes include, but are not limited to, RNAses, DNAses, proteases,kinases, and caspases. Suitable proteases are described in, e.g.,Bosslet et al. (1992) Br J Cancer 65:234, Goshom et al. (1993) CancerRes 53:2123, Rodrigues et al. (1995) Cancer Res 55:63, Michael et al.(1996) Immunotechnology 2:47, Haisma et al. (1998) Blood 92:184.Exemplary radioisotopes include, but are not limited to, ³²P and ¹²⁵I.Suitable radionuclides are also described in, e.g., Colcher et al.(1999) Ann N Y Acad Sci 880:263. Additional exemplary effector moietiesare, for example, Fc fragments from homologous or heterologousantibodies.

It will be appreciated by those of skill in the art that the sequence ofany protein effector molecule may be altered in a manner that does notsubstantially affect the functional advantages of the effector protein.For example, glycine and alanine are typically considered to beinterchangeable as are aspartic acid and glutamic acid and asparagineand glutamine. One of skill in the art will recognize that manydifferent variations of effector sequences will encode effectors withroughly the same activity as the native effector.

The effector molecule and the antibody may be conjugated by chemical orby recombinant means as described above. Chemical modifications include,for example, derivitization for the purpose of linking the effectormolecule and the antibody to each other, either directly or through alinking compound, by methods that are well known in the art of proteinchemistry. Both covalent and noncovalent attachment means may be usedwith the humanized antibodies of the present invention.

The procedure for attaching an effector molecule to an antibody willvary according to the chemical structure of the moiety to be attached tothe antibody. Polypeptides typically contain a variety of functionalgroups; e.g., carboxylic acid (COOH), free amine (—NH.sub.2) orsulfhydryl (—SH) groups, which are available for reaction with asuitable functional group on an antibody to result in the binding of theeffector molecule.

Alternatively, the antibody is derivatized to expose or to attachadditional reactive functional groups. The derivatization may involveattachment of any of a number of linker molecules such as thoseavailable from Pierce Chemical Company, Rockford Ill.

The linker is capable of forming covalent bonds to both the antibody andto the effector molecule. Suitable linkers are well known to those ofskill in the art and include, but are not limited to, straight orbranched-chain carbon linkers, heterocyclic carbon linkers, or peptidelinkers. Where the antibody and the effector molecule are polypeptides,the linkers may be joined to the constituent amino acids through theirside groups (e.g., through a disulfide linkage to cysteine). However, ina preferred embodiment, the linkers will be joined to the alpha carbonamino and carboxyl groups of the terminal amino acids.

In some circumstances, it is desirable to free the effector moleculefrom the antibody when the immunoconjugate has reached its target site.Therefore, in these circumstances, immunoconjugates will compriselinkages that are cleavable in the vicinity of the target site. Cleavageof the linker to release the effector molecule from the antibody may beprompted by enzymatic activity or conditions to which theimmunoconjugate is subjected either inside the target cell or in thevicinity of the target site. When the target site is a tumor, a linkerwhich is cleavable under conditions present at the tumor site (e.g. whenexposed to tumor-associated enzymes or acidic pH) may be used.

In the presently preferred chemical conjugation embodiment, the means oflinking the effector molecule and the antibody comprises aheterobifunctional coupling reagent which ultimately contributes toformation of an intermolecular disulfide bond between the effectormolecule and the antibody. Other types of coupling reagents that areuseful in this capacity for the present invention are described, forexample, in U.S. Pat. No. 4,545,985. Alternatively, an intermoleculardisulfide may conveniently be formed between cysteines in the effectormolecule and the antibody which occur naturally or are inserted bygenetic engineering. The means of linking the effector molecule and theantibody may also use thioether linkages between heterobifunctionalcrosslinking reagents or specific low pH cleavable crosslinkers orspecific protease cleavable linkers or other cleavable or noncleavablechemical linkages. The means of linking the effector molecule and theantibody may also comprise a peptidyl bond formed between the effectormolecule and the antibody which are separately synthesized by standardpeptide synthesis chemistry or recombinant means.

Exemplary chemical modifications of the effector molecule and theantibody of the present invention also include derivitization withpolyethylene glycol (PEG) to extend time of residence in the circulatorysystem and reduce immunogenicity, according to well known methods (Seefor example, Lisi, et al., Applied Biochem. 4:19 (1982); Beauchamp, etal., Anal Biochem. 131:25 (1982); and Goodson, et al., Bio/Technology8:343 (1990)).

Antibodies of the present invention may optionally be covalently ornon-covalently linked to a detectable label. Detectable labels suitablefor such use include any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. Useful labels in the present invention include magneticbeads (e.g. DYNABEADS), fluorescent dyes (e.g., fluoresceinisothiocyanate, Texas red, rhodamine, green fluorescent protein, and thelike), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g.,horse radish peroxidase, alkaline phosphatase and others commonly usedin an ELISA), and colorimetric labels such as colloidal gold or coloredglass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels may be detected using photographicfilm or scintillation counters, fluorescent markers may be detectedusing a photodetector to detect emitted illumination. Enzymatic labelsare typically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

The antibody and/or immunoconjugate compositions of this invention areparticularly useful for parenteral administration, such as intravenousadministration or administration into a body cavity.

The compositions for administration will commonly comprise a solution ofthe antibody and/or immunoconjugate dissolved in a pharmaceuticallyacceptable carrier, preferably an aqueous carrier. A variety of aqueouscarriers can be used, e.g., buffered saline and the like. Thesesolutions are sterile and generally free of undesirable matter. Thesecompositions may be sterilized by conventional, well known sterilizationtechniques. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiological conditionssuch as pH adjusting and buffering agents, toxicity adjusting agents andthe like, for example, sodium acetate, sodium chloride, potassiumchloride, calcium chloride, sodium lactate and the like. Theconcentration of fusion protein in these formulations can vary widely,and will be selected primarily based on fluid volumes, viscosities, bodyweight and the like in accordance with the particular mode ofadministration selected and the patient's needs.

Thus, a typical pharmaceutical composition of the present invention forintravenous administration would be about 0.1 to 10 mg per patient perday. Dosages from 0.1 up to about 100 mg per patient per day may beused. Actual methods for preparing administrable compositions will beknown or apparent to those skilled in the art and are described in moredetail in such publications as REMINGTON'S PHARMACEUTICAL SCIENCE, 19THED., Mack Publishing Company, Easton, Pa. (1995).

The compositions of the present invention can be administered fortherapeutic treatments. In therapeutic applications, compositions areadministered to a patient suffering from a disease, in an amountsufficient to cure or at least partially arrest the disease and itscomplications. An amount adequate to accomplish this is defined as a“therapeutically effective dose.” Amounts effective for this use willdepend upon the severity of the disease and the general state of thepatient's health. An effective amount of the compound is that whichprovides either subjective relief of a symptom(s) or an objectivelyidentifiable improvement as noted by the clinician or other qualifiedobserver.

Single or multiple administrations of the compositions are administereddepending on the dosage and frequency as required and tolerated by thepatient. In any event, the composition should provide a sufficientquantity of the proteins of this invention to effectively treat thepatient. Preferably, the dosage is administered once but may be appliedperiodically until either a therapeutic result is achieved or until sideeffects warrant discontinuation of therapy. Generally, the dose issufficient to treat or ameliorate symptoms or signs of disease withoutproducing unacceptable toxicity to the patient.

Controlled release parenteral formulations of the immunoconjugatecompositions of the present invention can be made as implants, oilyinjections, or as particulate systems. For a broad overview of proteindelivery systems see, Banga, A. J., THERAPEUTIC PEPTIDES AND PROTEINS:FORMULATION, PROCESSING, AND DELIVERY SYSTEMS, Technomic PublishingCompany, Inc., Lancaster, Pa., (1995) incorporated herein by reference.Particulate systems include microspheres, microparticles, microcapsules,nanocapsules, nanospheres, and nanoparticles. Microcapsules contain thetherapeutic protein as a central core. In microspheres the therapeuticis dispersed throughout the particle. Particles, microspheres, andmicrocapsules smaller than about 1 μm are generally referred to asnanoparticles, nanospheres, and nanocapsules, respectively. Capillarieshave a diameter of approximately 5 μm so that only nanoparticles areadministered intravenously. Microparticles are typically around 100 μmin diameter and are administered subcutaneously or intramuscularly. See,e.g., Kreuter, J., COLLOIDAL DRUG DELIVERY SYSTEMS, J. Kreuter, ed.,Marcel Dekker, Inc., New York, N.Y., pp. 219-342 (1994); and Tice &Tabibi, TREATISE ON CONTROLLED DRUG DELIVERY, A. Kydonieus, ed., MarcelDekker, Inc. New York, N.Y., pp. 315-339, (1992) both of which areincorporated herein by reference.

Polymers can be used for ion-controlled release of immunoconjugatecompositions of the present invention. Various degradable andnondegradable polymeric matrices for use in controlled drug delivery areknown in the art (Langer, R., Accounts Chem. Res. 26:537-542 (1993)).For example, the block copolymer, polaxamer 407 exists as a viscous yetmobile liquid at low temperatures but forms a semisolid gel at bodytemperature. It has shown to be an effective vehicle for formulation andsustained delivery of recombinant interleukin-2 and urease (Johnston, etal., Pharm. Res. 9:425-434 (1992); and Pec, et al., J. Parent. Sci.Tech. 44(2):58-65 (1990)). Alternatively, hydroxyapatite has been usedas a microcarrier for controlled release of proteins (Ijntema, et al.,Int. J. Pharm. 112:215-224 (1994)). In yet another aspect, liposomes areused for controlled release as well as drug targeting of thelipid-capsulated drug (Betageri, et al., LIPOSOME DRUG DELIVERY SYSTEMS,Technomic Publishing Co., Inc., Lancaster, Pa. (1993)). Numerousadditional systems for controlled delivery of therapeutic proteins areknown. See, e.g., U.S. Pat. Nos. 5,055,303, 5,188,837, 4,235,871,4,501,728, 4,837,028 4,957,735 and 5,019,369, 5,055,303; 5,514,670;5,413,797; 5,268,164; 5,004,697; 4,902,505; 5,506,206, 5,271,961;5,254,342 and 5,534,496, each of which is incorporated herein byreference.

As used herein, the terms “cancer” and “cancerous” refer to or describethe physiological condition in mammals in which a population of cellsare characterized by unregulated cell growth. Examples of cancerinclude, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma,leukemia, benign or malignant tumors. More particular examples of suchcancers include squamous cell cancer, small-cell lung cancer, non-smallcell lung cancer, adenocarcinoma of the lung, squamous carcinoma of thelung, cancer of the peritoneum, hepatocellular cancer, gastrointestinalcancer, pancreatic cancer, glioblastoma, cervical cancer, ovariancancer, liver cancer, bladder cancer, hepatoma, breast cancer, coloncancer, colorectal cancer, endometrial or uterine carcinoma, salivarygland carcinoma, kidney cancer, liver cancer, prostate cancer, vulvalcancer, thyroid cancer, brain, hepatic carcinoma and various types ofhead and neck cancer, neurofibromatosis type I or II. Other examples ofsuch cancers include those that are therapy resistant, refractory ormetastatic.

Other diseases or disorders which may be treated in include“inflammatory diseases or disorders.” “Inflammatory disease or disorder”as used herein include, and are not limited to, pruritis, skininflammation, psoriasis, multiple sclerosis, rheumatoid arthritis,osteoarthritis, systemic lupus erythematosus, Hashimoto's thyroidis,myasthenia gravis, diabetes type I or II, asthma, inflammatory lunginjury, inflammatory liver injury, inflammatory glomerular injury,atopic dermatitis, allergic contact dermatitis, irritant contactdermatitis, seborrhoeic dermatitis, Sjoegren's syndrome,keratoconjunctivitis, uveitis, inflammatory bowel disease, Crohn'sdisease, ulcerative colitis, an inflammatory disease of the joints,skin, or muscle, acute or chronic idiopathic inflammatory arthritis,myositis, a demyelinating disease, chronic obstructive pulmonarydisease, interstitial lung disease, interstitial nephritis and chronicactive hepatitis.

“Metastasis” as used herein refers to the process by which a cancerspreads or transfers from the site of origin to other regions of thebody with the development of a similar cancerous lesion at the newlocation. A “metastatic” or “metastasizing” cell is one that losesadhesive contacts with neighboring cells and migrates via thebloodstream or lymph from the primary site of disease to invadeneighboring body structures.

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to, humans, non-human primates,rodents, and the like, which is to be the recipient of a particulartreatment. Typically, the terms “subject” and “patient” are usedinterchangeably herein in reference to a human subject.

As used herein, the term “subject suspected of having cancer” refers toa subject that presents one or more symptoms indicative of a cancer(e.g., a noticeable lump or mass) or is being screened for a cancer(e.g., during a routine physical). A subject suspected of having cancercan also have one or more risk factors. A subject suspected of havingcancer has generally not been tested for cancer. However, a “subjectsuspected of having cancer” encompasses an individual who has receivedan initial diagnosis but for whom the stage of cancer is not known. Theterm further includes people who once had cancer (e.g., an individual inremission).

As used herein, the term “subject at risk for cancer” refers to asubject with one or more risk factors for developing a specific cancer.Risk factors include, but are not limited to, gender, age, geneticpredisposition, environmental exposure, previous incidents of cancer,preexisting non-cancer diseases, and lifestyle.

As used herein, the term “characterizing cancer in a subject” refers tothe identification of one or more properties of a cancer sample in asubject, including but not limited to, the presence of benign,pre-cancerous or cancerous tissue, the stage of the cancer, and thesubject's prognosis. Cancers can be characterized by the identificationof the expression of one or more cancer marker genes, including but notlimited to, the cancer markers disclosed herein.

As used herein, the terms “cell cancer marker(s)”, “cancer cellmarker(s)”, “tumor cell marker(s)”, or “solid tumor cell marker(s)”refer to a gene or genes or a protein, polypeptide, or peptide expressedby the gene or genes whose expression level, alone or in combinationwith other genes, is correlated with the presence of tumorigenic cancercells compared to non-tumorigenic cells, such as integrin β1. Thecorrelation can relate to either an increased or decreased expression ofthe gene (e.g. increased or decreased levels of mRNA or the peptideencoded by the gene).

As used herein, the term “detecting a decreased or increased expressionrelative to non-cancerous control” refers to measuring the level ofexpression of a gene (e.g., the level of mRNA or protein) relative tothe level in a non-cancerous control sample. Gene expression can bemeasured using any suitable method, including but not limited to, thosedescribed herein.

As used herein, the term “detecting a change in gene expression in acell sample in the presence of said test compound relative to theabsence of said test compound” refers to measuring an altered level ofexpression (e.g., increased or decreased) in the presence of a testcompound relative to the absence of the test compound. Gene expressioncan be measured using any suitable method.

As used herein, the term “instructions for using said kit for detectingcancer in said subject” includes instructions for using the reagentscontained in the kit for the detection and characterization of cancer ina sample from a subject.

As used herein, “providing a diagnosis” or “diagnostic information”refers to any information that is useful in determining whether apatient has a disease or condition and/or in classifying the disease orcondition into a phenotypic category or any category having significancewith regards to the prognosis of or likely response to treatment (eithertreatment in general or any particular treatment) of the disease orcondition. Similarly, diagnosis refers to providing any type ofdiagnostic information, including, but not limited to, whether a subjectis likely to have a condition (such as a tumor), information related tothe nature or classification of a tumor as for example a high risk tumoror a low risk tumor, information related to prognosis and/or informationuseful in selecting an appropriate treatment. Selection of treatment caninclude the choice of a particular chemotherapeutic agent or othertreatment modality such as surgery or radiation or a choice aboutwhether to withhold or deliver therapy.

As used herein, the terms “providing a prognosis”, “prognosticinformation”, or “predictive information” refer to providing informationregarding the impact of the presence of cancer (e.g., as determined bythe diagnostic methods of the present invention) on a subject's futurehealth (e.g., expected morbidity or mortality, the likelihood of gettingcancer, and the risk of metastasis).

As used herein, the term “post surgical tumor tissue” refers tocancerous tissue (e.g., biopsy tissue) that has been removed from asubject (e.g., during surgery).

As used herein, the term “subject diagnosed with a cancer” refers to asubject who has been tested and found to have cancerous cells. Thecancer can be diagnosed using any suitable method, including but notlimited to, biopsy, x-ray, blood test, and the diagnostic methods of thepresent invention.

As used herein, the terms “biopsy tissue”, “patient sample”, “tumorsample”, and “cancer sample” refer to a sample of cells, tissue or fluidthat is removed from a subject for the purpose of determining if thesample contains cancerous tissue, including cancer cells or fordetermining gene expression profile of that cancerous tissue. In someembodiment, biopsy tissue or fluid is obtained because a subject issuspected of having cancer. The biopsy tissue or fluid is then examinedfor the presence or absence of cancer, cancer cells, and/or cancer cellgene signature expression.

In certain embodiments of the present invention, cancer cell expression,such as pancreatic, colon, breast, liver, kidney, brain, GBM and thelike, comprises elevated levels of integrin β1 compared tonon-tumorigenic colon tumor cells. Integrins are heterodimericextracellular matrix (ECM) cell-surface proteins that consist of both analpha and a beta chain with chains associating with multiple partners toform different integrins. Integrins function in cellular adhesion andmigration to reversibly connect cells to the extracellular matrix or toreceptors on other cells and thus can play a critical role in cancerinvasion and metastasis. Integrin-mediated adhesion also affectsintracellular signaling and can thus regulate cell survival,proliferation, and differentiation.

Integrin beta 1 can form functional receptors with the largest diversityof known alpha integrins, resulting in the ability to interact with adiverse range of ECM environments, and has been implicated in cancer.For example, increased beta 1 integrin signaling is associated withmalignant progression of breast cancer both clinically and in breastcancer cell lines.

The integrin β1 has been identified as directly effecting tumor growth.Specifically, treatment of tumor cells with anti-integrin β1 antibodiesreduces tumor size and inhibits metastasis.

In some embodiments, the present invention provides methods fordetection of expression of integrin β1 as a marker for cancer. In someembodiments, expression is measured directly (e.g., at the proteinlevel), in some embodiments, expression is detected in tissue samples(e.g., biopsy tissue). In other embodiments, expression is detected inbodily fluids (e.g., including but not limited to, plasma, serum, wholeblood, mucus, and urine). The present invention further provides kitsfor the detection of markers, in some embodiments, the presence of acell cancer marker is used to provide a prognosis to a subject. Theinformation provided is also used to direct the course of treatment. Forexample, if a subject is found to have a marker indicative of a solidtumor cell, additional therapies (e.g., hormonal or radiation therapies)can be started at an earlier point when they are more likely to beeffective (e.g., before metastasis). In addition, if a subject is foundto have a tumor that is not responsive to hormonal therapy, the expenseand inconvenience of such therapies can be avoided.

In embodiments, gene expression of integrin β1 is detected by measuringthe level of the protein. Protein expression can be detected by anysuitable method. In some embodiments, proteins are detected byimmunohistochemistry utilizing the antibodies described herein.

Antibody binding is detected by techniques known in the art (e.g.,radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitationreactions, immunodiffusion assays, in situ immunoassays (e.g., usingcolloidal gold, enzyme or radioisotope labels, for example), Westernblots, precipitation reactions, agglutination assays (e.g., gelagglutination assays, hemagglutination assays, etc.), complementfixation assays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc.

In one embodiment, antibody binding is detected by detecting a label onthe primary antibody. In another embodiment, the primary antibody isdetected by detecting binding of a secondary antibody or reagent to theprimary antibody. In a further embodiment, the secondary antibody islabeled. Many methods are known in the art for detecting binding in animmunoassay and are within the scope of the present invention.

In some embodiments, an automated detection assay is utilized. Methodsfor the automation of immunoassays include those described in U.S. Pat.Nos. 5,885,530, 4,981,785, 6,159,750, and 5,358,691, each of which isherein incorporated by reference. In some embodiments, the analysis andpresentation of results is also automated. For example, in someembodiments, software that generates a prognosis based on the presenceor absence of a series of proteins corresponding to cancer markers isutilized.

In other embodiments, the immunoassay described in U.S. Pat. Nos.5,599,677 and 5,672,480, each of which is herein incorporated byreference, can be used.

In some embodiments, a computer-based analysis program is used totranslate the raw data generated by the detection assay (e.g., thepresence, absence, or amount of a given marker or markers) into data ofpredictive value for a clinician. The clinician can access thepredictive data using any suitable means. Thus, in some embodiments, thepresent invention provides the further benefit that the clinician, whois not likely to be trained in genetics or molecular biology, need notunderstand the raw data. The data is presented directly to the clinicianin its most useful form. The clinician is then able to immediatelyutilize the information in order to optimize the care of the subject.

The present invention contemplates any method capable of receiving,processing, and transmitting the information to and from laboratoriesconducting the assays, information providers, medical personal, andsubjects. For example, in some embodiments of the present invention, asample (e.g., a biopsy or a serum or urine sample) is obtained from asubject and submitted to a profiling service (e.g., clinical lab at amedical facility, genomic profiling business, etc.), located in any partof the world (e.g., in a country different than the country where thesubject resides or where the information is ultimately used) to generateraw data. Where the sample comprises a tissue or other biologicalsample, the subject can visit a medical center to have the sampleobtained and sent to the profiling center, or subjects can collect thesample themselves and directly send it to a profiling center. Where thesample comprises previously determined biological information, theinformation can be directly sent to the profiling service by the subject(e.g., an information card containing the information can be scanned bya computer and the data transmitted to a computer of the profilingcenter using an electronic communication system). Once received by theprofiling service, the sample is processed and a profile is produced(e.g., expression data), specific for the diagnostic or prognosticinformation desired for the subject.

The profile data is then prepared in a format suitable forinterpretation by a treating clinician. For example, rather thanproviding raw expression data, the prepared format can represent adiagnosis or risk assessment for the subject, along with recommendationsfor particular treatment options. The data can be displayed to theclinician by any suitable method. For example, in some embodiments, theprofiling service generates a report that can be printed for theclinician (e.g., at the point of care) or displayed to the clinician ona computer monitor.

In some embodiments, the information is first analyzed at the point ofcare or at a regional facility. The raw data is then sent to a centralprocessing facility for further analysis and/or to convert the raw datato information useful for a clinician or patient. The central processingfacility provides the advantage of privacy (all data is stored in acentral facility with uniform security protocols), speed, and uniformityof data analysis. The central processing facility can then control thefate of the data following treatment of the subject. For example, usingan electronic communication system, the central facility can providedata to the clinician, the subject, or researchers.

In some embodiments, the subject is able to directly access the datausing the electronic communication system. The subject can chose furtherintervention or counseling based on the results. In some embodiments,the data is used for research purposes. For example, the data can beused to further optimize the inclusion or elimination of markers asuseful indicators of a particular condition or stage of disease.

In yet other embodiments, the present invention provides kits for thedetection and characterization of cancer. In some embodiments, the kitscontain antibodies specific for a cancer marker, such as the antibodiesof the present invention, in addition to detection reagents and buffers.In other embodiments, the kits contain reagents specific for thedetection of mRNA or cDNA (e.g., oligonucleotide probes or primers). Insome embodiments, the kits contain all of the components necessaryand/or sufficient to perform a detection assay, including all controls,directions for performing assays, and any necessary software foranalysis and presentation of results.

Another embodiment of the present invention comprises a kit to test forthe presence of proteins such as integrin β1, e.g. in a tissue sample orin a body fluid. The kit can comprise, for example, an antibody fordetection of a polypeptide. In addition, the kit can comprise areference or control sample; instructions for processing samples,performing the test and interpreting the results; and buffers and otherreagents necessary for performing the test.

In some embodiments, in vivo imaging techniques are used to visualizethe expression of cancer markers in an animal (e.g., a human ornon-human mammal). For example, in some embodiments, integrin β1 islabeled using a labeled antibody of the present invention. Aspecifically bound and labeled antibody can be detected in an individualusing an in vivo imaging method, including, but not limited to,radionuclide imaging, positron emission tomography, computerized axialtomography, X-ray or magnetic resonance imaging method, fluorescencedetection, and chemiluminescent detection.

The in vivo imaging methods of the present invention are useful in thediagnosis of cancers that express the solid tumor cell cancer markers ofthe present invention (e.g., in breast cancer). In vivo imaging is usedto visualize the presence of a marker indicative of the cancer. Suchtechniques allow for diagnosis without the use of an unpleasant biopsy.The in vivo imaging methods of the present invention are also useful forproviding prognoses to cancer patients. For example, the presence of amarker indicative of cancer cells can be detected. The in vivo imagingmethods of the present invention can further be used to detectmetastatic cancers in other parts of the body.

In some embodiments, reagents (e.g., antibodies) specific for the cancermarkers of the present invention are fluorescently labeled. The labeledantibodies are introduced into a subject (e.g., orally or parenterally).Fluorescently labeled antibodies are detected using any suitable method(e.g., using the apparatus described in U.S. Pat. No. 6,198,107, hereinincorporated by reference).

In other embodiments, antibodies are radioactively labeled. The use ofantibodies for in vivo diagnosis is well known in the art.

In some embodiments, the present invention provides therapies fordiseases and disorders including immunological/inflammatory diseases anddisorders given the role of integrin β1 in broader functional activitiesas discussed above. Further, diseases and disorders which may betargeted by the compositions of the present invention include multiplesclerosis, Crohn's disease, rheumatoid arthritis, inflammatory boweldisease and the like. Similarly, it is to be expected that certain eyerelated diseases may be targeted including wet age-related maculardegeneration (AMD).

In some embodiments, the present invention provides therapies fordiseases such as cancer, e.g., breast, brain, prostate and colon cancer.In some embodiments, therapies target cancer markers, such as integrinβ1.

In some embodiments, the present invention provides antibodies thattarget tumors that express a cell cancer marker, e.g., integrin β1.

In some embodiments, the therapeutic antibodies comprise an antibody ofthe present invention conjugated to a cytotoxic agent as discussedabove.

The following example is provided to further illustrate the advantagesand features of the present invention, but are not intended to limit thescope of the invention. While they are typical of those that might beused, other procedures, methodologies, or techniques known to thoseskilled in the art may alternatively be used.

Example 1 Variable Region Gene Sequencing

Genes encoding the OS2966 anti-human integrin β1 monoclonal antibodywere subjected to variable (V)-region sequence analysis. Total RNA wasextracted from 3 to 10×10⁶ hybridoma cells using the RNAqueous-4PCR Kit™(Ambion, Warrington, UK) and used to synthesize cDNA. Murineimmunoglobulin heavy and kappa light chain V-region fragments wereamplified by PCR using degenerate mouse leader sequence primers (Sigma)and unique constant domain primers (Sigma) as shown in Table 1. Theresulting PCR fragments were subcloned into the pGEM-T Easy I™ vectorsystem (Promega, Southampton, UK) and inserts were sequenced using thevector-specific primer, M13Forward (Sigma) All DNA sequencing wasperformed by Geneservice Ltd, Cambridge, UK). Unique V-region nucleotidesequences were obtained for OS2966 (SEQ ID NOs: 1 (VH) and 3 (VL)).

TABLE 1 SEQ ID Sequence Name-Pool NO ATGRASTTSKGGYTMARCTKGRTTTMuIgV_(H)5′-A 58 ATGRAATGSASCTGGGTYWTYCTCTT MuIgV_(H)5′-B 59ATGGACTCCAGGCTCAATTTAGTTTTCCT MuIgV_(H)5′-C 60ATGGCTGTCYTRGBGCTGYTCYTCTG MuIgV_(H)5′-C 61ATGGVTTGGSTGTGGAMCTTGCYATTCCT MuIgV_(H)5′-C 62ATGAAATGCAGCTGGRTYATSTTCTT MuIgV_(H)5′-D 63 ATGGRCAGRCTTACWTYYTCATTCCTMuIgV_(H)5′-D 64 ATGATGGTGTTAAGTCTTCTGTACCT MuIgV_(H)5′-D 65ATGGGATGGAGCTRTATCATSYTCTT MuIgV_(H)5′-E 66 ATGAAGWTGTGGBTRAACTGGRTMuIgV_(H)5′-E 67 ATGGRATGGASCKKIRTCTTTMTCT MuIgV_(H)5′-E 68ATGAACTTYGGGYTSAGMTTGRTTT MuIgV_(H)5′-F 69 ATGTACTTGGGACTGAGCTGTGTATMuIgV_(H)5′-F 70 ATGAGAGTGCTGATTCTTTTGTG MuIgV_(H)5′-F 71ATGGATTTTGGGCTGATTTTTTTTATTG MuIgV_(H)5′-F 72 CCAGGGRCCARKGGATARACIGRTGGMuIgGV_(H)3′-2 73 ATGRAGWCACAKWCYCAGGTCTTT MuIgkV_(L)5′-A 74ATGGAGACAGACACACTCCTGCTAT MuIgkV_(L)5′-B 75ATGGAGWCAGACACACTSCTGYTATGGGT MuIgkV_(L)5′-C 76ATGAGGRCCCCTGCTCAGWTTYTTGGIWTCTT MuIgkV_(L)5′-D 77TGGGCWTCAAGATGRAGTCACAKWYYCWGG MuIgkV_(L)5′-D 78ATGAGTGTGCYCACTCAGGTCCTGGSGTT MuIgkV_(L)5′-E 79ATGTGGGGAYCGKTTTYAMMCTTTTCAATTG MuIgkV_(L)5′-E 80ATGGAAGCCCCAGCTCAGCTTCTCTTCC MuIgkV_(L)5′-E 81ATGAGIMMKTCIMTTCAITTCYTGGG MuIgkV_(L)5′-F 82 ATGAKGTHCYCIGCTCAGYTYCTIRGMuIgkV_(L)5′-F 83 ATGGTRTCCWCASCTCAGTTCCTTG MuIgkV_(L)5′-F 84ATGTATATATGTTTGTTGTCTATTTCT MuIgkV_(L)5′-F 85ATGAAGTTGCCTGTTAGGCTGTTGGTGCT MuIgkV_(L)5′-G 86ATGGATTTWCARGTGCAGATTWTCAGCTT MuIgkV_(L)5′-G 87ATGGTYCTYATVTCCTTGCTGTTCTGG MuIgkV_(L)5′-G 88ATGGTYCTYATVTTRCTGCTGCTATGG MuIgkV_(L)5′-G 89 ACTGGATGGTGGGAAGATGGAMuIgkV_(L)3′-1 90

Example 2 Generation of Chimeric Antibodies

The heavy and light chain variable domain sequences of the OS2966monoclonal antibody were PCR amplified and subcloned into pANT antibodyexpression vectors (FIG. 14) with heavy and light chain V-regions clonedinto pANT17 and pANT13 respectively. Heavy chain V-region genes werecloned into pANT17 via MluI and HindIII sites in frame with either thehuman χ1 heavy chain gene (G1m3 (G1m(f)) allotype) or the human χ4 heavychain gene, and light chain V-region genes were cloned into pANT13 viaBssHII and BamHI sites in frame with the human kappa light chainconstant region gene (Km3 allotype). Transcription of both heavy andlight chain genes was under the control of the CMV I/E promoter (U.S.Pat. No. 5,168,062 and U.S. Pat. No. 5,385,839, University of Iowa) andthe pANT17 plasmid contained a mutant dhfr minigene (Simonsen & Levinson1983, PNAS 80:2495-2499) under the control of a SV40 promoter and polyAsequence for selection in eukaryotic cells. Both pANT17 and pANT13contained a β-lactamase (Ap^(R)) gene for prokaryotic selection and apMB1 origin of replication for propagation in prokaryotic cells. Allplasmids were propagated in E. coli XL1-blue (Stratagene Cat. No.200130).

The heavy and light chain expression constructs were then co-transfectedeither transiently into HEK293 cells by calcium phosphate-basedtransfection or stably transfected into NS0 cells by electroporation.Secreted antibody was purified from the cell culture supernatants byProtein A chromatography.

Example 3 Generation of Humanized Antibodies

Humanized antibodies were generated using methods described in EP1844074(Antitope Ltd). Structural models of the mouse V-regions were producedusing Swiss PDB and analyzed in order to identify important amino acidsfrom the OS2966 V-regions that were likely to be important for theintegrin β1 binding properties of the antibody (constraining residues').A database of human V-region sequences was used to identify segments ofhuman V-region sequences containing each of the constraining residues tobe used in design of the humanized antibodies. Typically two or morealternative V-region sequence segments were used to provide eachconstraining residue resulting in a large range of possible sequences ofhumanized anti-integrin B1 V-region sequences. These sequences were thenanalysed for the prediction of non-germline MHC class II peptide bindingby in silico analysis as described in Fothergill et al. (WO9859244,assignee Eclagen Ltd) and also for known CD4+ T-cell epitopes usingdatabases including “The Immune Epitope Database and Analysis Resource”,available on the world wide web at URL: immuneepitope.org/. V-regionsequences with predicted non-germline MHC class II binding peptides, orwith significant hits against T cell epitope databases were discarded.This resulted in a reduced set of V-region sequences. Selectedcombinations of V-region sequence segments were then combined to producehumanized heavy and light chain variable region amino acid sequences.Five heavy chains and four light chain sequences (designated VH1 to VH5,and Vκ1 to Vκ4 respectively) were selected (SEQ ID NOs: 6, 8, 10, 12,14, 16, 18, 20, 22) for gene synthesis. In addition, a further set ofheavy and light chain V-region sequences (designated VH6 to VH15, andVκ5 to Vκ13 respectively) were designed (SEQ ID NOs: 29 to 38 and 44 to52 respectively).

In addition to the above, humanized antibodies were designed using thegeneral methods of Winter (U.S. Pat. No. 6,548,640B2) as modified inWinter, Carr, Harris (EP0629240B1) whereby sequences were designed usingthe CDR sequences of OS2966 (SEQ ID NOs: 23 to 28) to replace CDRs in aset of human germline V-region sequences with addition of human J regionsequences. In addition, constraining residues as used in the humanizedantibodies of the above paragraph were introduced into the germlineV-region framework sequences. The resultant sequences for the heavy andlight chain V-regions were designated VH16 to VH20, and Vκ14 to Vκ18respectively, and are listed as SEQ ID NOs: 39-43 and 53-57.

In addition to the above, deimmunised antibodies were designed using thegeneral methods of Carr et al. (U57465572 B2) whereby CD4+ T cellepitopes within the V-region sequences of OS2966 (SEQ ID NOs: 2 and 4)were identified and mutations introduced in order to potentially removeone or more of these epitopes.

DNA encoding humanized OS2966 variant V-regions were synthesized andsubcloned into the expression vectors pANT17 and pANT13 as described inExample 2. All combinations of humanized VH and Vκ chains (i.e.combinations of VH1 to VH5, and Vκ1 to Vκ4, (i.e. a total of 20pairings) were transiently transfected into HEK293 and also transfectedinto NS0 cells, and antibody was purified by protein A chromatographyfrom the culture supernatants as described in Example 2.

Example 4 Analysis of Humanized Antibodies

The binding of HEK-derived and NS0-derived OS2966 humanized variants tohuman integrin β1 was assessed in a competition ELISA against thechimeric antibody from Example 2. The OS2966 chimeric antibody wasbiotinylated using Biotin Tag™ Micro Biotinylation kit (Sigma-Aldrich).96 well MaxiSorp plates (Nunc) were coated with 0.5 μg/ml human integrinβ1 (100 μl final volume) at 4° C. overnight. The plates were washed withwash buffer (0.05% Tween20 in Dulbecco's-PBS) and blocked withDulbecco's PBS-2% BSA for 1 hour at room temperature. Plates were thenwashed 3 times with wash buffer. Test humanized antibodies at variousconcentrations were premixed with biotinylated chimeric antibody (0.02μg/ml final concentration) and then added to the human integrin(31-coated plate (100 μl final volume). Plates were incubated for 1 h atroom temperature and washed 3 times with wash buffer. 100 μl of a 1 in500 dilution of Streptavidin HRP (Sigma-Aldrich) was added and incubatedfor 1 hour at room temperature. Plates were washed 3 times with washbuffer and 100 μl of SigmaFast OPD substrate (Sigma-Aldrich, Cat# P9187)was added and incubated at room temperature in the dark for 4 minutes.The reaction was stopped by adding 50 μl of 3M HCl. Plates were read at490 nm using Dynex plate reader.

Example 5 Generation of scFv's and Fab's

Humanized OS2966 anti-human integrin variants from Example 3 wereconverted into scFv's and cloned into M13 phage display vectors asdescribed in Benhar I. and Reiter Y., Current Protocols in Immunology,Unit 10.19B, Wiley Online Library, May 2002 (available on the world wideweb at URL: currentprotocols.com/protocol/im1019b) using the pCANTAB5Evector RPAS Expression Module (Amersham Pharmacia Biotech, LittleChalfont, UK). Humanized VH and VK genes were amplified using primerswhich provided terminal SfiI and NotI restriction sites, an internalGly4Ser linker and a C terminal his6 tag. The scFv constructs wereinserted into the pCANTAB5E vector as SfiI-NotI fragments andtransformed into E. coli HB2151 resulting in scFv exported to theperiplasm and partially to the growth medium. scFv's were purified fromgrowth medium by nickel-chelate affinity chromatography using HIS-SelectHF Cartridges (Sigma-Aldrich). Purified scFv's were tested in thecompetition assay as detailed in Example 4. Humanized OS2966 variantsfrom Example 3 were also converted into Fab's using the method used forscFv's except that amplified humanized VH and VK genes were furtheramplified with CH1 and Cκ constant region genes to form VH-CH1 and VK-Cκfragments which were further amplified with primers to join thesefragments with a 22 amino acid pelB leader sequence (Lei S. P., Lin H.C., Wang S. S., Callaway J., and Wilcox G., J. Bacteriol. 169 (1987) p4379-4383) between the upstream VH-CH1 and downstream VK-Cκ genefragments resulting in a dicistronic Fab gene. Fab's from humanizedantibody variants were generated and purified as above for scFv's andtested in the human integrin β1 competition assay as detailed in Example4.

Example 6 Analysis of CD4+ T Cell Responses

The immunogenicity potential of the humanized antibodies from Examples 3and 5 was performed in comparison to the humanized anti-human integrinβ1 K20 (Poul et al., Molecular Immunology 32 (1995) p 102-116). PBMCs(peripheral blood mononuclear cells) were isolated from healthycommunity donor buffy coats (from blood drawn within 24 hours) obtainedfrom the UK National Blood Transfusion Service (Addenbrooke's Hospital,Cambridge, UK) and according to approval granted by Addenbrooke'sHospital Local Research Ethics Committee. PBMCs were isolated from buffycoats by Lymphoprep (Axis-shield, Dundee, UK) density centrifugation andCD8⁺ T cells were depleted using CD8⁺ RosetteSep™ (StemCell TechnologiesInc, London, UK). Donors were characterized by identifying HLA-DRhaplotypes using an HLA SSP-PCR based tissue-typing kit (Biotest,Solihull, UK). T cell responses to control antigens including the recallantigen tetanus toxin were also determined (KLH Pierce, Cramlingtom, UKand peptides derived from Influenza A and Epstein Barr viruses). PBMCwere then frozen and stored in liquid nitrogen until required.

To prepare monocyte derived dendritic cells (DC), 50 different donorPBMCs were selected to provide a distribution with frequencies of HLA-DRand HLA-DQ allotypes similar to the frequencies in the overall worldpopulation. PBMCs were revived in AIM-V® culture medium and CD14⁺ cellsisolated using Miltenyi CD14 Microbeads and LS columns (MiltenyiBiotech, Oxford, UK). Monocytes were resuspended in AIM-V® supplementedwith 1000 U/ml IL-4 and 1000 U/ml GM-CSF (“DC culture media”) to 4-6×10⁶PBMC/ml and then distributed in 24 well plates (2 ml final culturevolume). Cells were fed on day 2 by half volume DC culture media change.By day 3, monocytes had differentiated to semi-mature DC which werepre-incubated with either 40 ug/ml of test humanized or chimericantibody, 100 μg/ml KLH or media only. Semi-mature DC were incubatedwith antigen for 24 hours after which excess test antibody was removedby washing the cells twice and resuspending in DC culture mediasupplemented with 50 ng/ml TNF-α (Peprotech, London, UK). DCs were fedon day 7 by a half volume DC culture media (supplemented with 50 ng/mlTNFα) change before harvesting mature DC on day 8. The harvested matureDC were counted and viability assessed using trypan blue dye exclusion.The DC were then γ-irradiated (4000 rads) and resuspended at 2×10⁵ cellsper ml in AIM-V media before use in the ELISpot and proliferationassays. Additionally, on day 8, fresh CD4+ T cells were also prepared.To purify CD4+ T cells, PBMCs were revived in AIM-V® culture medium andCD4⁺ cells isolated using Miltenyi CD4 Microbeads and LS columns(Miltenyi Biotech, Oxford, UK) and resuspended in AIM-V® media at 2×10⁶cells/ml.

On day 8, T cell proliferation assays were established whereby 1×10⁵autologous CD4⁺ T cells were added to 1×10⁴ humanized or chimericantibody loaded DC (ratio of 10:1) in 96 well U-bottomed plates, withAIM-V® media added to a final volume 200 ul/well). On day 14, assayplates were pulsed with 1 uCi [3H] (Perkin Elmer, Beaconsfield, UK) perwell in 25 ul AIMV for 6 hours before harvesting onto filter mats(Perkin Elmer) using a TomTec Mach III (Hamden Conn., USA) cellharvester. All antibody preparations were tested in sextuplet cultures.Counts per minute (cpm) for each well were determined by Meltilex™(Perkin Elmer) scintillation counting on a 1450 Microbeta Wallac TriluxLiquid Scintillation Counter (Perkin Elmer) in paralux, low backgroundcounting. Counts per minute for each antibody sample were normalized tothe media only control.

For ELISpot assays, ELISpot plates (Millipore, Watford, UK) were coatedwith 100 ul/well IL-2 capture antibody (R&D Systems, Abingdon, UK) inPBS. Plates were then washed twice in PBS, incubated overnight in blockbuffer (1% BSA (Sigma) in PBS) and washed in AIM V® medium. On day 8,1×10⁵ autologous CD4⁺ T cells were added to 1×10⁴ antigen loaded DC(ratio of 10:1) in 96 well ELISpot plates. All antibody preparationswere tested in sextuplet cultures. For each donor PBMC, a negativecontrol (AIM V® medium alone), no cells control and a PHA (10 ug/ml)positive control were also included.

After a further 7 day incubation period, ELISpot plates were developedby three sequential washes in dH₂O and PBS prior to the addition of 100ulfiltered biotinylated detection antibody (R&D Systems, Abingdon, UK)in PBS/1% BSA. Following incubation at 37° C. for 1.5 hour, plates werefurther washed three times in PBS and 100 ul filtered streptavidin-AP(R&D Systems) in PBS/1% BSA was added for 1 hour (incubation at roomtemperature). Streptavidin-AP was discarded and plates were washed fourtimes in PBS. BCIP/NBT (R&D Systems) was added to each well andincubated for 30 minutes at room temperature. Spot development wasstopped by washing the wells and the backs of the wells three times withdH₂O. Dried plates were scanned on an Immunoscan™ Analyser and spots perwell (spw) were determined using Immunoscan™ Version 4 software.

For both proliferation and IL-2 ELISpot assays, results were expressedas a Stimulation Index (SI) defined as the ratio of cpm (proliferationassay) or spots (ELISpot assay) for the test antibody against amedium-only control using a threshold of SI equal to or greater than 2(SI≥2.0) for positive T cell responses.

Example 7 Tumor Animal Model

A tumor animal model can be used for the in vivo analysis of humanizedanti-integrin 131 antibodies in inhibiting tumor growth. For example, anorthotic breast cancer model using MDA-MB-231 cells may be used as amodel of primary tumor growth and spontaneous metastasis in humanintegrin β1 knock-in mice or immune deficient mice (e.g., nu/nu, severecombined immune deficiency [SCID]).

Integrin β1 knock-in mice (7-10 weeks old, males and females distributedequally across groups) may be injected subcutaneously into the mammaryfat pad with MDA-MB-231 cells in 0.1 ml volume. Anti-integrin β1antibody of the present invention, OS2966 or an isotype matched controlantibody may be injected at 1 mg/kg-20 mg/kg, such as 5 mg/kg or 10mg/kg doses (dosing volume 10 ml/kg) weekly starting the day followingtumor cell administration (“Day 2”) or when the tumor is palpable andmean tumor volume is approximately 100 mm³. Tumor measurements may betaken biweekly during the course of the experiment by calipermeasurement. Animals may be followed to determine results.

Example 8 Sequence Analysis

Sequence analysis was performed on the heavy and light chains generatedin the Examples to determine important VH and VL amino acid residuesoutside of CDRs from the OS2966 sequence which are preferably includedin the humanized sequences. Such residues are identical to those ofOS2966 in addition to those of CDRs. Such residues are identified as “c”residues in FIGS. 15 and 16. As shown, for the VH chain residues 48, 67,69, 73, 76, 80, 89, 91 and 93 are preferably identical to correspondingresidues of OS2966 (SEQ ID NO:2). Also, for the VL chain, residues 36and 71 are preferably identical to corresponding residues of OS2966 (SEQID NO:4).

Codon usage corresponding to the VH and VL sequences is provided in FIG.17 while FIG. 18 provides a summary of the amino acid sequences of theVH and VL chains.

Example 9 Method of Increasing Radiation Sensitivity by Inhibition ofIntegrin β1

Humanized antibodies of the present invention may be utilized toincrease radiation sensitivity by inhibition of integrin β1 as disclosedin U.S. Patent Appl. Pub. No. 20070237711, Park et al., Cancer Res 2008;68:(11)4398, and Yao et al., Cancer Res 2007; 67:(2)659, all of whichare incorporated herein in their entireties. The antibodies of thepresent invention may be utilized in a method of co-administration ofthe antibody or antibody containing compositions described herein, incombination with ionizing radiation that causes increased apoptosis intumor cells, notably in breast cancer tumor cells.

Example 10 Validation and Analysis of Composite Human Antibody Variants

Composite humanized antibodies were generated as discussed in Example 3.Table 2 provides a list of the various antibodies generated showingwhich VH and Vk sequences were utilized.

TABLE 2 Variant designations according to V region ID. HUMANIZEDCORRESPONDING VH VARIANT REF. NO. V REGION ID AND VK SEQ ID NOS. H1 VH5VK3 14, 20 H2  VH3VK1 10, 16 H3  VH4VK4 12, 22 H4  VH5VK4 14, 22 H5 VH5VK2 14, 18 H6  VH5VK1 14, 16 H7  VH4VK3 12, 20 H8  VH4VK2 12, 18 H9 VH4VK1 12, 16 H10 VH3VK4 10, 22 H11 VH3VK3 10, 20 H12 VH3VK2 10, 18 H13VH2VK4  8, 22 H14 VH2VK3  8, 20 H15 VH2VK2  8, 18 H16 VH2VK1  8, 16 H17VH1VK3  6, 20 H18 VH1VK2  6, 18 H19 VH1VK1  6, 16

Binding of composite human antibody variants (from Table 2) was assessedin a competition FACS assay with the OS2966 murine chimeric antibody(murine OS2966 Fab sliced to human Fc). In brief, a dilution series(three-fold) of chimeric or humanized antibodies starting from 25.0μg/ml was premixed with a constant concentration of murine OS2966(0.1875 μg/ml) in FACS buffer (1% BSA in 1×PBS pH 7.4) before mixingwith 3×10⁵ Jurkat cells. After incubating on ice for 1 hour, cells werewashed and the binding of murine OS2966 was detected with PE labelledgoat anti-murine Fc (Jackson ImmunoResearch, Cat. No. 115-116-071).After incubating on ice for 45 min, cells were washed, resuspended in300 μl FACS buffer and analyzed on a Beckton Dickinson FACScalibur™. Thegeometric mean fluorescence intensity was plotted against antibodyconcentration (FIGS. 20-24). These data were used to calculate IC50values for each antibody and these values were normalized to the IC50 ofchimeric antibody that was included in each FACS assay (Table 3).

TABLE 3 Human variant relative affinity. The relative IC50 wascalculated by dividing the IC50 of the test antibody by that of thechimeric antibody assayed in the same assay. VARIANT RELATIVE AFFINITY(IC50) H1  0.88 H2  0.97 H3  2.70 H4  3.26 H5  0.85 H6  1.24 H7  1.76H8  1.18 H9  1.34 H10 2.76 H11 1.58 H12 1.15 H13 2.00 H14 1.17 H15 1.32H16 1.08 H17 1.35 H18 1.20 H19 1.13

Three variants (H1, H2, H5) demonstrated slightly greater relativeaffinities and four variants (H3, H4, H10, H13) had significantly lowerrelative affinities than murine OS2966 antibody.

Functional inhibition of the integrin β1 subunit with the human variantantibodies was assessed in an adhesion microplate assay on multiple ECMand in multiple cancer cell lines PANC-1 human pancreatic cancer (FIGS.25A and 25B), MDA-MB-231 human triple negative breast cancer (FIG. 25C),and AsPC-1 human pancreatic cancer (FIG. 25D). In brief, ECM adhesionassays were performed with 10 ECM components or BSA (control). Cellswere pre-incubated with 10 μg/ml antibodies in serum-free DMEM/F12 mediafor 30 min Adhesion was tested for 30 min to 1 hr. Non-adherent cellswere removed from dishes, cells fixed with 1% paraformaldehyde, andplates stained with crystal violet for 30 min. After extensive rinsing,crystal violet was solubilized in Triton X-100 for 1 hr and plates readfor absorbance at A590.

All 19 human variants were functionally active in attenuating celladhesion to all ECM with some significant variation depending on ECMprotein and cell line. Relative affinities (Table 3) do not demonstrateobvious correlation to function in this assay regardless of ECM type orcell line. These data were normalized to IgG negative control (BSA,bovine serum albumin as negative control; IgG, isotype control; OS2966,murine OS2966; TS2/16, positive control integrin β1 activating antibody;FN, fibronectin; LN, laminin; C1, collagen type I; C4, collagen typeIV).

Functional inhibition of the integrin β1 subunit with the human variantantibodies was assessed in a microplate “scratch wound” migration assayon ECM component fibronectin with human triple negative breast cancercells (MDA-MB-231). Briefly, plates were coated with 10 μg/mlfibronectin and seeded with tumor cells. A yellow pipette tip was usedto scratch monolayers of confluent cells and media replaced including 10μg/ml of stated antibodies. Plates were fixed after 8 hr incubation at37 C and imaged. 10× magnification images of plates demonstrateattenuation of migration into the wound in OS2966 and composite humanvariant (H1, H2, H3) treated wells (FIG. 26A). Quantitation of cell freearea for each condition (performed in triplicate and repeated) is shownin FIG. 26B.

Migration of triple negative breast cancer cells was significantlyattenuated by treatment with OS2966 composite human variants.

Functional inhibition of the integrin β1 subunit with the human variantantibodies was assessed in an in vitro model of angiogenesis; the tubeforming assay with human umbilical vein endothelial cells (HUVEC).Briefly, plates were coated with Matrigel ECM and seeded with HUVECcells. Plates were imaged after 8 hr incubation at 37 C and quantitatedfor vascular tube formation (closed unit formation). 10× magnificationimages of plates demonstrate attenuation of vascular tube formation inOS2966 and composite human variant (H1, H2, H3) treated wells (FIG.27A). Quantitation of closed unit formation for each condition(performed in at least triplicate and repeated with endothelialprogenitor cells) is shown in FIG. 27B.

The composite human variants completely block angiogenesis in the invitro tube forming assay.

Functional inhibition of the integrin β1 subunit with the human variantantibodies was assessed in an in vivo model of human triple negativebreast cancer with the MDA-MB-231 cell line. Briefly, 10⁶ cells wereinjected into the 4^(th) mammary fat pad in 5- to 6-week old female nudeathymic nu/nu mice. Mice were randomized into treatment groups aftertumors were established (mean subcutaneous volume=80-100 mm³). Controland experimental antibodies were administered at 5 mg/kgintraperitoneally (IP) twice weekly. Human IgG at equivalent dosesserved as the control (Sigma). Orthotopic tumors were measured withcalipers twice weekly. As shown in FIG. 28A, human variants H1-H3significantly attenuated growth of MDA-MB-231 tumors in vivo compared toIgG control. A trend towards superior efficacy for the composite humanvariants was apparent compared to murine OS2966, particularly for H3.Pharmacodynamic Western Blot analysis demonstrated a reduction ofactivities in critical pro-growth signaling pathways (phosphorylatedExtracellular-related Kinase, ERK and phosphorylated Focal AdhesionKinase, FAK) in the treated tumors compared to control which may havecontributed to reduced proliferation and elevation of apoptosis (FIG.28B).

Functional inhibition of the integrin β1 subunit with the human variantantibodies was assessed in an in vivo model of spontaneous lungmetastasis with human triple negative breast cancer with the MDA-MB-231cell line. Briefly, 10⁶ cells were injected into the 4^(th) mammary fatpad in 5- to 6-week old female nude athymic nu/nu mice. Mice wererandomized into treatment groups after tumors were established (meansubcutaneous volume=80-100 mm³). Control and experimental antibodieswere administered at 5 mg/kg intraperitoneally (IP) twice weekly. HumanIgG at equivalent doses served as the control (Sigma). After 7 weeks,mice were perfused, lungs collected, and sectioned coronally for H&Eanalysis. Spontaneous lung metastasis was observed in 50% (3 of 6) ofmice given IgG control antibody. Spontaneous lung metastases were notobserved in any mice given OS2966 or composite human variants H1-H3 (0%,0/28 mice). Results are shown in FIG. 29.

Composite human variants completely prevented spontaneous lungmetastasis in an orthotopic model of triple negative breast cancer.

Functional inhibition of the integrin β1 subunit with the human variantantibodies was assessed in an in vivo model of human gemcitabineresistant pancreatic cancer with the PANC1-GEMR cell line. Briefly, 10⁶cells were injected subcutaneously in 5- to 6-week old male nude athymicnu/nu mice. Mice were randomized into treatment groups after tumors wereestablished (mean subcutaneous volume=80-100 mm³). Control andexperimental antibodies were administered at 5 mg/kg intraperitoneally(IP) twice weekly or gemcitabine at 50 mg/kg. Human IgG at equivalentdoses served as the control (Sigma). Subcutaneous tumors were measuredwith calipers twice weekly. FIG. 30A shows in vivo verification ofgemcitabine resistance in the PANC1-GEMR line. FIG. 30B shows thatcomposite human variant H3 significantly attenuated growth of PANC1-GEMRtumors in vivo compared to IgG control. FIG. 30C shows PharmacodynamicWestern Blot analysis of critical pro-growth signaling pathways(phosphorylated Extracellular-related Kinase, ERK and phosphorylatedFocal Adhesion Kinase, FAK) in the treated tumors compared to controlwhich may have contributed to reduced proliferation and elevation ofapoptosis.

Functional inhibition of the integrin β1 subunit with the human variantantibodies was assessed in an in vivo model of established humanglioblastoma with the U87MG cell line. Briefly, 10⁷ cells were injectedsubcutaneously in 5- to 6-week old male nude athymic nu/nu mice. Micewere randomized into treatment groups after tumors were well established(mean subcutaneous volume=˜200-250 mm³). Control and experimentalantibodies were administered at 5 mg/kg intraperitoneally (IP) twiceweekly. Human IgG at equivalent doses served as the control (Sigma).Subcutaneous tumors were measured with calipers twice weekly. FIG. 31Ademonstrates that composite human variant H3 significantly attenuatedgrowth of established glioblastoma tumors in vivo compared to IgGcontrol. Efficacy of H3 was equivalent to murine OS2966. PharmacodynamicWestern Blot analysis demonstrated a reduction of activities in criticalpro-growth signaling pathways (phosphorylated Extracellular-relatedKinase, ERK and phosphorylated AkT) in the H3 treated tumors compared tocontrol which may have contributed to reduced proliferation andelevation of apoptosis as shown in FIG. 31B.

The EpiScreen™ time course T cell proliferation assay was used todetermine the potential for clinical immunogenicity of antibody variantH3. The fully humanized and chimeric antibodies were tested for theirability to induce CD4⁺ T cell responses as measured by proliferationagainst a panel of 12 HLA-typed donors. Healthy donor T cellproliferation responses to the chimeric (murine/human) OS2966 antibody,humanized H3 antibody and a positive control humanized antibody areshown in FIG. 32A. CD4+ T cells were incubated with autologous mature DCloaded with the samples and assessed for proliferation after 7 daysincubation. Proliferation responses with an SI≥2.00 (indicated by reddotted line) that were significant (p<0.05) using an unpaired, twosample student's t test were considered positive. FIG. 32B provides asummary of healthy donor T cell proliferation responses to the donorcohort. Positive (SI≥2.00, significant p<0.05) T cell responses forproliferation (“P”) are shown. Borderline responses (significant p<0.05with SI≥1.90) are shown (*). The frequency of positive responses for theproliferation assay are shown as a percentage at the bottom of thecolumns. A33 (humanized A33) is the clinical benchmark control mAb thatshows high levels of immunogenicity in the clinic and routinely induces20-30% T cell responses in the EpiScreen assay. For each donor, animmunogenic reproducibility control (cells incubated with 100 μg/ml KLH)was also included.

The composite human variant H3 demonstrated significantly reducedimmunogenicity (0% responses) compared to the OS2966 murine/humanchimera (25% responses). It is concluded that the human antibody H3exhibits a clinically acceptable immunogenicity profile from theEpiScreen™ assay providing confirmation of reduced immunogenicity as aresult of the Antitope Composite Human Antibody technology.

Although the invention has been described with reference to the aboveexample, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

What is claimed is:
 1. A humanized antibody which specifically binds integrin β1 comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region have amino acid sequences as set forth in SEQ ID NOs:6 and 16, SEQ ID NOs:6 and 18, SEQ ID NOs:6 and 20, SEQ ID NOs:8 and 16, SEQ ID NOs:8 and 18, SEQ ID NOs:8 and 20, SEQ ID NOs:8 and 22, SEQ ID NOs:10 and 16, SEQ ID NOs:10 and 18, SEQ ID NOs:10 and 20, SEQ ID NOs:10 and 22, SEQ ID NOs:12 and 16, SEQ ID NOs:12 and 18, SEQ ID NOs:12 and 20, SEQ ID NOs:12 and 22, SEQ ID NOs:14 and 16, SEQ ID NOs:14 and 18, SEQ ID NOs:14 and 20, and SEQ ID NOs:14 and
 22. 2. The antibody of claim 1, wherein CDRs of the VH and VL regions are from a donor antibody.
 3. The antibody of claim 1, wherein the antibody comprises an Fc region.
 4. The antibody of claim 3, wherein the Fc region is of IgG1, IgG2, IgG3, or IgG4.
 5. The antibody of claim 4, wherein the Fc region is a human IgG1 or IgG4.
 6. The antibody of claim 1, wherein the antibody comprises a light chain constant region.
 7. The antibody of claim 6, wherein the light chain constant region is of isotype kappa.
 8. The antibody of claim 1, wherein the antibody is an scFv or Fab.
 9. The antibody of claim 1, wherein the antibody is a humanized antibody.
 10. The antibody of claim 1, wherein the antibody is a chimeric antibody.
 11. The antibody of claim 1, wherein the antibody binds integrin β1 with an equilibrium dissociation constant (Kd) of at least 10⁻²M, 10⁻³M, 10⁻⁴M, 10⁻⁵M, 10⁻⁶M, 10⁻⁷M, 10⁻⁸M, 10⁻⁹M or 10⁻¹⁰M.
 12. The antibody of claim 1, which when tested in vitro for induction of CD4+ helper T cell responses in blood samples with a distribution of HLA-DR allotypes from the human population, gives rise to less than 10% of T cell responses.
 13. The antibody of claim 12, which when tested gives rise to less than 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of T cell responses.
 14. A multi-specific antibody comprising an antibody of claim
 1. 15. A pharmaceutical composition comprising the antibody of claim 1 and a pharmaceutically acceptable carrier.
 16. An immunoconjugate comprising the antibody of claim 1 linked to a detectable or therapeutic moiety.
 17. The immunoconjugate of claim 16, wherein the therapeutic moiety is a cytotoxic moiety.
 18. The immunoconjugate of claim 16, wherein the therapeutic moiety is a chemotherapeutic agent.
 19. The immunoconjugate of claim 16, wherein the detectable moiety is a fluorescent moiety.
 20. The antibody of claim 1, wherein the VH region and the VL region have amino acid sequences as set forth in SEQ ID NOs:10 and 16, SEQ ID NOs:12 and 22, or SEQ ID NOs:14 and
 20. 21. The antibody of claim 1, wherein the VH region and the VL region have amino acid sequences as set forth in SEQ ID NOs:12 and
 22. 